Small molecule based antibody-recruiting compounds for cancer treatment

ABSTRACT

The present invention relates to chimeric (including bifunctional) compounds, compositions comprising those compounds and methods of treating cancer in a patient or subject, especially including metastatic cancer where cancer cells exhibit overexpression (heightened expression) of cell surface urokinase-type plasminogen activator receptor (urokinase receptor) compared to normal (non-cancerous) cells. The compounds bind to the urokinase-type plasminogen activator receptor (uPAR) on the surface of a cancer cell, including a metastatic cancer cell, and consequently recruit native antibodies of the patient or subject where the antibodies can selectively degrade and/or deactivate targeted cancer cells through antibody-dependent cellular phagocytosis and antibody-dependent cellular cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) against a large number and variety of cancers, thus providing cancer cell death and an inhibition of growth, elaboration and/or metastasis of the cancer, including remission and cure of the patient&#39;s cancer.

RELATED APPLICATIONS AND GRANT SUPPORT

This application claims the benefit of priority of U.S. provisionalapplication Nos. 62/201,812, filed Aug. 6, 2015 and 62/290,793, filedFeb. 3, 2016, both applications entitled “Small Molecule BasedAntibody-Recruiting Agent Targeting uPAR”, the entire contents of saidapplications being incorporated by reference herein.

This invention was made with government support under 1DP2OD002913-01awarded by the National Institute of Health. The government has certainrights in the invention.

FIELD OF THE INVENTION

The present invention relates to chimeric (including bifunctional)compounds, compositions comprising those compounds and methods oftreating cancer in a patient or subject, especially including metastaticcancer and other cancers where cancer cells exhibit overexpression(heightened expression) of cell surface urokinase-type plasminogenactivator receptor (urokinase receptor) compared to normal(non-cancerous) cells. The compounds bind to the urokinase-typeplasminogen activator receptor (uPAR) on the surface of a cancer cell,including a metastatic cancer cell, and consequently recruit nativeantibodies of the patient or subject where the antibodies canselectively remove, destroy, clear and/or deactivate targeted cancercells through antibody-dependent cellular phagocytosis (ADCP),antibody-dependent cellular cytotoxicity (ADCC), complement dependentcytotoxicity (CDC) and/or other immune effector mechanisms against alarge number and variety of cancers, thus providing cancer cell deathand an inhibition of growth, elaboration and/or metastasis of thecancer, including remission and cure of the patient's cancer.

INTRODUCTION/BACKGROUND OF THE INVENTION

Cancer is currently the second leading cause of death in the UnitedStates. Metastatic cancers are especially difficult to treat and areassociated with higher levels of mortality compared to benign tumors.American men and women have a 38-44% chance of developing invasivecancers over the course of their lifetimes. Tumor metastasis involvescancer cell invasion of surrounding tissues, often accelerated by cellsurface proteases. One such protease known as the urokinase-typeplasminogen activator (uPA) is capable of breaking down extracellularmatrix proteins and activating migration-inducing signal cascadesthrough binding to the urokinase-type plasminogen activator receptor(uPAR).

A large body of evidence suggests that uPA and uPAR expression aresubstantially higher on invasive malignant cancer cells than on healthycells or benign tumors. In clinical settings, high levels of uPAR areused as diagnostic measures for metastatic potential and poor clinicaloutcome in several malignancies. Novel strategies to combat cancer arehighly desirable due to the limitations of the more traditionaltreatment options, including radiation therapy and chemotherapy. Thesetreatment methods are not only associated with significant side effectsbut are also limited with respect to their effectiveness in thetreatment of late stage cancers.

The ability to target cancer cells selectively is thus of greatimportance, with the potential to significantly reduce toxicity andoff-target effects, thereby reducing side effects experienced by thepatient. New approaches to treat cancer that combine the advantages oftraditional small molecules and biologics could address many of thelimitations associated with currently available therapies.

Anthroquinone-based small molecules have been identified previously aspotential anti-cancer agents and have been shown to bind to uPAR invitro with triple digit nM affinity capable of blocking cancer cellinvasion, migration, and adhesion at double to triple uM concentrations(T. Mani et al). Previously, the development of an antineoplasticantibody-recruiting agent equipped with the urokinase protein as a uPARtarget-binding domain was reported from the present laboratory. Themolecule developed here, termed “ARM-U,” was shown previously toselectively target uPAR-over-expressing A172 glioblastoma cells andfacilitate anti-DNP antibody-dependent cellular phagocytosis (ADCP) atsingle digit nM concentrations. Although ARM-U has high-affinity bindingto uPAR (K_(d) of ˜200 pM), its potential as a therapeutic is limiteddue to the incorporation of the large uPA protein with limited stabilityin vivo. This, plus the limitations associated with the administrationof uPA protein limits its use.

The present invention describes the rational design of the highestaffinity small molecule targeting the uPA site on uPAR ever reported.Novel small molecule derived ARM-U2 compounds demonstrate efficacyagainst metastatic cancer cells in cellular assays at low nMconcentrations. Moreover, these compounds do not require theadministration of uPA protein in order to facilitate its therapeuticefficacy, a clear advance over the prior art compounds.

The present invention also illuminates the nature of the non-covalentinteractions with the uPA binding site on uPAR required for tightbinding to uPAR. In addition, the present invention has providedexperimentally supported computational predictions regarding theimportance of basic residues in the uPA binding pocket for targetingsmall molecules selectively to uPAR and reports the first crystalstructure of a uPAR targeting antibody recruiting small molecule bindingthe uPA binding site of uPAR. An additional feature of the presentinvention is that it demonstrate the high potency and efficacy of anantibody recruiting small molecule.

The present invention shows that (ARM-U2) binds uPAR on the surface ofA172 glioblastoma and B16 melanoma cancer cells and recruits endogenousanti-DNP antibodies to the cell surface which in turn induce antibodydependent cellular phagocytosis and antibody dependent cellularcytotoxicity (see FIG. 1). Present preliminary in-vivo data show theability of ARM-U2 to inhibit tumor formation using a B-16 xenograftmouse tumor model

BRIEF DESCRIPTION OF THE INVENTION

The present invention is directed to compounds according to the generalchemical structure:

-   Where    is a moiety which binds to an active site of urokinase-type    plasminogen activator receptor (uPAR) on the surface of cancer cells    of a patient or subject;-   is an antibody binding moiety comprising a hapten which is capable    of binding to an antibody in said patient or subject (preferably an    endogenous antibody which pre-exists in the patient or subject    without having to be raised prior to therapy);-   Each L1 is a linker molecule which chemically links    to CT, L2 or    in said compound;-   Each L2 is a linker molecule which chemically links    to CT, L1 or    in a molecule;-   Each CT is independently an optional connector molecule which, when    present links L1 or L2 to    , L1 or L2 to    and L1 to L2;-   Each j is independently 0, 1, 2, 3, 4 or 5 (preferably 0 or 1, more    preferably 1);-   Each k is independently 0, 1, 2, 3, 4 or 5 (preferably 0 or 1, more    preferably 1), with the proviso that at least one CT is present when    k and j are both 0 (preferably at least one of k and j is 1); and-   Each m and n is independently an integer from 1 to 15, 1 to 10, 1 to    5, 1 to 3, 2 to 3, 2 to 5, 1 to 2 or 1 (often m is 1 and n is 1-6,    more often 1, 2, 3 or 4),-   or a pharmaceutically acceptable salt, solvate or polymorph thereof.

In certain embodiments, compounds according to the present invention arerepresented by the chemical structure:

-   Wherein each R^(N) is independently H or a C₁-C₃ alkyl group when N    is an amine group or each R^(N) is absent when N forms an isoxazole    group by binding to the adjacent oxygen atom;-   R₁, R₂, R₃, R₄ and R₅ are each independently H, a halogen (F, Cl,    Br, I, preferably F), a C₁-C₃ alkyl group optionally substituted    with one or two hydroxyl groups or up to three fluoro groups, NO₂,    CN, a (CH₂)_(m′)OR^(E) (O-alkyl) group, a (CH₂)_(m′)COR^(E) (keto)    group, a (CH₂)_(m′)COOR^(E) (carboxy ester) group, a (CH₂)_(m′)SO₃H    group, a (CH₂)_(m′)OCOR^(E) (oxycarbonyl ester) group,

-   Each R′ is independently H or a C₁-C₃ alkyl group (preferably H or    CH₃, most often H);-   R_(a) is a sidechain derived from a natural or unnatural amino acid    (D- or L-, preferably a L-amino acid) preferably selected from the    group consisting of alanine (methyl), arginine (propyleneguanidine),    asparagine (methylenecarboxyamide), aspartic acid (ethanoic acid),    cysteine (thiol, reduced or oxidized di-thiol), glutamine    (ethylcarboxyamide), glutamic acid (propanoic acid), histidine    (methyleneimidazole), isoleucine (1-methylpropane), leucine    (2-methylpropane), lysine (butyleneamine), methionine    (ethylmethylthioether), phenylalanine (benzyl), proline (R′ forms a    cyclic ring with R_(a) and the adjacent nitrogen group to form a    pyrrolidine group), hydroxyproline, serine (methanol), threonine    (ethanol, 1-hydroxyethane), tryptophan (methyleneindole), tyrosine    (methylene phenol) or valine (isopropyl)-   Each R^(E) is H or a C₁-C₆ alkyl group optionally substituted with    one or two hydroxyl groups or up to three chloro or fluoro groups    (preferably R^(E) is H or a C₁-C₃ alkyl group);-   R_(1′), R_(2′), R_(3′) R_(4′) and R_(5′) are each independently H, a    halogen (F, Cl, Br, I, preferably F), a C₁-C₆ (preferably C₁-C₃)    alkyl group optionally substituted with one or two hydroxyl groups    or up to three chloro or fluoro groups, NO₂, CN, a (CH₂)_(m′)OR^(E)    (O-alkyl) group, a (CH₂)_(m′)COOR^(E) (carboxy ester) group, a    (CH₂)_(m′)O—COR^(E) (oxycarbonyl ester) group or a (CH₂)_(m′)COR^(E)    (keto) group;-   Each m′ is independently 0, 1, 2, 3, 4, 5, or 6 (preferably 0, 1, 2    or 3, more preferably 0 or 1);-   Each y′ is independently 0, 1 or 2 (preferably 0 or 1);-   R^(LABT) is an

-   where L is a bond, at least one linker (preferably a single linker)    which comprises a first linker group L1 which optionally includes a    connector group CT and an optional linker group L2 which itself    optionally includes a connector group CT, said first linker group L1    being linked to said second linker group L2 optionally (preferably)    through a CT group; and-   is an antibody binding moiety comprising a hapten which is capable    of binding to an antibody in said patient or subject, or-   a pharmaceutically acceptable salt, stereoisomer, enantiomer,    solvate or polymorph thereof.

In preferred embodiments compounds according to the present invention isrepresented by the chemical structure:

Where the substituents on the compound R′, R_(1′), R_(2′), R_(3′),R_(4′), R_(5′), R₁, R₂, R₃, R₄, R₅ and R^(LABT) are the same as for thegeneric compound above or an enantiomer thereof.

In other preferred embodiments, compounds according to the presentinvention are represented by the chemical structure:

Where the substituents R^(N), R′, R_(1′), R_(2′), R_(3′), R_(4′),R_(5′), R₁, R₂, R₃, R₄, R₅ and R^(LABT) are the same as for the genericcompound above.

In preferred embodiments of the invention, R₁ is H, CO₂H or SO₃H; R₂ isH, CO₂H, SO₃H, —NHCH₂—CO₂H, —NHCH₂—SO₃H, —C(O)—NHCH₂—CO₂H or—C(O)—NHCH₂—SO₃H; R₃ is H, CO₂H, SO₃H, —NHCH₂—CO₂H, —NHCH₂—SO₃H,—C(O)—NHCH₂—CO₂H or —C(O)—NHCH₂—SO₃H (preferably H, CO₂H or SO₃H); R₄ isH, SO₃H or CO₂H (preferably H or CO₂H; R⁵ is H, SO₃H or CO₂H (preferablyH) and

-   R′, R_(1′), R_(2′), R_(3′), R_(4′), R_(5′) are H,-   or a pharmaceutically acceptable salt or solvate thereof.

In other preferred embodiments of the invention,

is

-   where X_(L) is N(R¹), O, S, S(O), SO₂, S(O)₂O, —OS(O)₂, or OS(O)₂O    (preferably N(R¹) or O, more preferably N(R¹)); and-   R¹ is H, a C₁-C₃ alkyl group or a —C(O)(C₁-C₃) group, preferably H;-   each n and n′ is independently 1 to 25, 1 to 15, 1 to 12, 2 to 11, 2    to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4 and 2 to 3 or 1, 2, 3, 4, 5,    6, 7, 8 or 9; and-   each n″ is independently 0 to 8, often 1 to 7, or 1, 2, 3, 4, 5 or 6    (preferably 3 or 6).

In certain preferred embodiments, L, L1 and/or L2 are (poly)ethyleneglycol groups comprising from 1 to 25, from 1 to 15, from 1 to 12, from2 to 11, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4 and 2 to 3ethyleneglycol groups, which may be linked to

, CT and/or

groups. In certain preferred embodiments of the invention, the connectorgroup CT, when present, is a group as otherwise described herein,including a triazole group, an amide group (which may contain one ormore methylene groups on either side of the amide), an alkylene group, asuccinimide group, or a diamide group according to the chemicalstructure

-   where R¹ is H or a C₁-C₃ alkyl group (preferably H) and n″ is 0 to    8, often 1 to 7, or 1, 2, 3, 4, 5 or 6 (preferably 3). Note is the    fact that the CT group may contain methylene groups of varying    lengths which connect the CT group to    , the linker group and/or    .

In certain embodiments, the

group is one or more dinitrophenyl (DNP) or rhamnose groups as describedin greater detail herein.

Preferred compounds according to the present invention are presented inattached FIG. 4, Scheme 1, FIG. 6, Table 1, FIG. 7 and FIG. 8 (withreference to FIG. 4) hereof.

In preferred embodiments of the present invention are directed tocompounds according to chemical structures:

-   Wherein R₁, R₂, R₃ and R₄ are each independently H, CO₂H, SO₃H,    CONHCH₂CO₂H or NHCH₂CO₂H;

-   n is 1-15, preferably 2-10, more often 3 or 7;-   n′ is 1-10, preferably=1-7, more often 2-6, often 3 or 6;-   ABT is a DNP group or a rhamnose group, often a DNP group; or-   a pharmaceutically acceptable salt, stereoisomer (a diastereomer or    enantiomer), solvate or polymorph thereof.

In another preferred embodiment, the present invention is directed to acompound according to the chemical structure:

-   Wherein-   R₁, R₃ and R₄ are each H and R₂ is CO₂H; or-   R₁ and R₃ are each H and R₂ and R₄ are each CO₂H; or-   R₁, R³ and R₄ are each H and R₂ is CONHCH₂CO₂H or NHCH₂CO₂H; or-   R₁, R₃ and R₄ are each H and R₂ is SO₃H; or-   R₁ and R₃ are each SO₃H and R₂ and R₄ are each H;-   X is

-   DNP is a

-    group; or-   a pharmaceutically acceptable salt or stereoisomer (a diastereomer    or enantiomer)

Additional preferred embodiments of compounds according to the presentinvention are presented in FIG. 7 hereof, including an enantiomer ordiastereomer thereof.

In additional embodiments of the invention, a pharmaceutical compositioncomprises an effective amount of a compound as described above,optionally and preferably in combination with a pharmaceuticallyacceptable carrier, additive or excipient. In alternative aspects,pharmaceutical combination compositions comprise an effective amount ofa compound as described herein, in combination with at least oneadditional agent which is used to treat cancer, including metastaticcancer, or a secondary condition or effect of cancer, especiallymetastatic cancer or alternative secondary effect, including one or moreof bone pain, hyperplasia, osteoporosis, kidney failure, liver failure,etc., as otherwise described herein.

In a further aspect of the invention, compounds according to the presentinvention are used to treat cancer in a patient. The method of treatingcancer comprises administering to a patient in need an effective amountof a compound as otherwise described herein in combination with apharmaceutically acceptable carrier, additive or excipient, optionallyin further combination with at least one additional agent which iseffective in treating cancer, including metastatic cancer, or one ormore of its secondary conditions or effects. The method of treatment maybe combined with alternative treatments, such as radiation therapy,among others.

The present invention also relates to a method for inhibiting cancer toreduce the likelihood or inhibit the spread or metastasis of the cancerinto other tissues of the patients' body for any cancer, and especiallysuch cancers including bone, lymph (lymph nodes), bladder, vas deferens,kidneys, liver, lungs, pancreas, brain, prostate and ovaries, amongothers.

Pursuant to the present invention, synthetic compounds for controllingor creating human immunity pursuant to the present invention have thepotential to revolutionize cancer treatment. Motivated by challenges inthis arena, the present inventors provide a strategy to targetmetastatic cancer cells for immune-mediated destruction by targeting theurokinase-type plasminogen activator receptor (uPAR). Urokinase-typeplasminogen activator (uPA) and uPAR are overexpressed on the surfacesof a wide range of invasive cancer cells and are believed to contributesubstantially to the migratory propensities of these cells. The keycomponent of the approach is an antibody-recruiting molecule thattargets the urokinase receptor (ARM-U). This bifunctional construct isformed by selectively, covalently attaching an antibody-binding smallmolecule to the active site of the urokinase enzyme (uPA) to produceARM-U2 compounds. The present inventors demonstrate that ARM-U2 iscapable of redirecting antibodies to the surfaces of target cancer cellsand mediating both antibody-dependent cellular phagocytosis (ADCP) andantibody-dependent cellular cytotoxicity (ADCC) against multiple humancancer cell lines. The present invention represents a novel technologyhas significant potential to impact the treatment of a variety ofdeadly, invasive cancers.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic representation of the ARM-U2 concept. In thefigure,

is represented as a TBT group and

is represented as a ABT group.

FIG. 2 shows computational docking studies of parent compound ARM-U21-ABT-1 in the uPA binding site of uPAR illustrating both theinteraction between 1-ABT-1 and uPAR hotspot residues as well as thesolvent exposed positioning of the

group.

FIG. 3 shows the crystal structure of ARM-U2 6-ABT-1 complexed to UPARat the UPA binding site making a key contract with Arg-53 through itssulfonate groups.

FIG. 4, Scheme 1 presents a chemical scheme for synthesizing a number ofARM-U2 compounds according to the present invention. General reactionconditions are as follows: i. NaNO₂/H2SO4 ii. NaN₃/H₂O iii. Toluenereflux iv. Methyl-3-aminobenzoate or, Dimethyl 5-aminoisophthalate,AlCl₃/PhNO₂ v. ABT-1, 2 or 3, DIPEA/DMF then NaOH/THF/MeOH 1:1:1 vi.1-ABT-1, GlyOme, HATU, DIPEA/DMF, then NaOH/THF/MeOH 1:1:1 vii.3-bromoaniline, GlyOMe, CuI, N,N-diethylsalicylamide, K₃PO₄/DMF viii.AlCl₃/PhNO₂ ix. ABT-1, DIPEA/DMF, then NaOH/THF/MeOH 1:1:1 x. metanillicacid, DIPEA/PhNO₂, or aniline-2,4-disulfonic acid, Li₂CO₃, Cu(OAc)₂/DMFxi. ABT-1, 2 or 3, DIPEA/DMF. Note that the (poly)ethylene glycollinkers depicted can range from 1 to 15 ethylene glycol units, often 2to 12 ethylene glycol units, often 2 to 10 ethylene glycol units, often3 to 9 ethylene glycol units, 3 to 8 ethylene glycol units or 3 to 7ethylene glycol units. The diamide connector group CT depicted can alsorange in size as otherwise described in the present application.

FIG. 5 shows the synthesis of a representative compound according to thepresent invention.

FIG. 6 shows a chemical synthetic scheme for a number of compoundsaccording to the present invention.

FIG. 7 shows a number of representative compounds according to thepresent invention. Note that for each of these compounds, the isoxazolecompounds are shown. Alternatively, these compounds may be readilyconverted to the ring-opened compounds (containing a keto group andamine group in place of the isoxazole group). See the examples sectionof the present application. Not all enantiomers are shown.

FIG. 8, Table 1 shows the chemical structures of a number ofrepresentative ARM-US compounds along with their calculated uPAR bindingaffinity (as determined by ELISA).

FIG. 9 A/B shows the dependence of 6-ABT-1 antibody recruitingcapability on cell surface uPAR illustrated using B16 melanoma cancercells either overexpressing (A) or failing to express uPAR (C) The ADCPefficacy exhibited by 6-ABT-1 on uPAR expressing A-172 glioblastomacells exhibiting a bell shaped dependence on 6-ABT-1 concentrationcharacteristic of the prozone effect. Key features of this data includethe significant increase in efficacy between 10 nM and 20 nM 6-ABT-1 inaccordance with its low double digit nM affinity for uPAR, the abilityto disrupt phagocytosis by out competition with exogenous uPAR andpyrimidine analog 6-ABT-4 and the complete loss of efficacy at 4 degreescelcius with 6-ABT-1 when phagocytosis is unable to occur D. Amnisimages of complete engulfment and cellular phagocytosis of A172 cells byu937 effector cells (indicated by co-localization of two cell stains)induced by 6-ABT-1 deemed positive for phagocytosis by the ADCP flowcytometry assay shown in C at 50 nM 6-ABT-1.

FIG. 10 shows in vivo efficacy studies of ARM-US 6-ABT-1 in an allograftmouse B16 tumor model. Black-line PBS administered control cohort, otherlines indicate ARM-U2 (at two different concentrations) or doxorubicinadministered (ip, 1 mpk) cohort.

FIG. 11 demonstrates the in vitro efficacy and potency of 6-ABT-1 inimmune effector cell assays using A172 glioblastoma cells. (A) 6-ABT-1concentration-dependent phagocytosis of A172 glioblastoma cells in thepresence of anti-DNP antibodies (133 nM). Studies were conducted at both37° C. or 4° C. and in the presence or absence of either exogenous uPARor derivative 20. (B) 6-ABT-1 induces enhanced release of inflammatorycytokine IL-8 from U937 cells accompanying ADCP assays at both 50 nM and1 μM compound concentrations.

FIG. 12 shows (A) Tumor growth inhibition in a B16 mouse melanomaallograft model expressing human uPAR. Tumor growth is measured over thecourse of several days upon treatment with PBS, doxorubicin at 1 mpk,combined doxorubicin (1 mpk)/ARM-U2 (6-ABT-1) (20 mpk) treatment, andtreatment with ARM-U2 (6-ABT-1) at 20 mpk and 100 mpk in mice graftedwith uPAR expressing B16 melanoma cells. (B) Kaplain-Meier curvesdemonstrating the prolongation of survival of mice allografted withhuman uPAR-positive tumors upon treatment with ARM-U2 (6-ABT-1) at both20 mpk and 100 mpk doxrubicin, or doxorubicin/ARM-U2 combinationcompared to mice treated with PBS. Dox=Doxorubicin, M.S=Median Survival(days). (c) Measured weight loss associated with treatment using ARM-U2(6-ABT-1) or doxorubicin.

FIG. 13 shows (A-B) Fluorescence titration of 100 nM candidate ARM-Uderivatives with increasing concentrations of uPAR accompanied by asaturatable quenching of ARM-U2 intrinsic fluorescence. (A). 1-ABT-1,275 nM+/−43 nM (B). 6-ABT-1, K_(d)=8.7 nM+/−3.3 nM, 6-ABT-2, K_(d)=66nM+/−9.5 nM, 6-ABT-3, 18 nM+/−5 nM. The generation of binding isothermsenabled for the calculation of solution equilibrium dissociationconstants for the binding interaction between select ARM-U2 derivativeswith uPAR. Extraction of K_(D) for each derivative was carried out usingthe quadratic equation S1 described in the examples section confirmingthe significantly increased affinity of 6-ABT-1 for uPAR compared to1-ABT-1. (C) Direct ELISA binding assay demonstrating the ability 2 tobind immobilized uPAR with much higher affinity than parent derivative 11-ABT-1. uPAR binding is also specific for the uPA binding site asdemonstrated by the displacement of 6-ABT-1 from uPAR following theaddition of exogenous 100 nM uPA-amino terminal fragment (ATF). (D)Competitive ELISA binding assay demonstrating the ability of the PEG-3linker 9 substituted ARM-U2 derivatives to compete with immobilized uPAfor uPAR. IC₅₀ abstracted from this data was used to calculate theinhibitory constant of each ARM-U2 derivative for uPAR employingequations 2 and 3. Highest affinity derivative was for 6-ABT-1.

FIG. 14 shows A. direct ELISA binding assay demonstrating the ability ofARM-U2 6-ABT-1 and 6-ABT-2 to bind immobilized uPAR with much higheraffinity than parent derivative 1-ABT-1. uPAR binding is also specificfor the uPA binding site as demonstrated by the displacement of 6-ABT-1from uPAR following the addition of exogenous 100 nM uPA-ATF. B.Competitive ELISA binding assay demonstrating the ability of the ABT-1ARM-U2 derivatives to compete with immobilized uPA for uPAR. IC50abstracted from this data was used to calculate the dissociationconstant of each ARM-U2 derivative for uPAR employing equations 1 and 2.Highest affinity derivative 6-ABT-1 shown in red trace. C. CompetitiveELISA binding isotherms for the longer linker (ABT-2/3) ARM-U2derivatives in addition to the (D)-stereoisomer of derivative 1-ABT-1.

FIG. 15 shows flow cytometric-antibody recruiting analysis of theability of ARM-U2 derivatives to bind simultaneously to both cellsurface uPAR and exogenously added anti-DNP antibodies. Top left panelshows the background antibody recruiting taking place in the presence ofA172 cells and anti-DNP antibody without the addition of ARM-U2. Middlepanel shows the antibody recruiting capability of 10 uM ARM-U2 1-ABT-1represented as a dot plot with the lower left gate representingbackground antibody recruiting in the absence of compound and the lowerright gate representing compound dependant antibody recruiting. Rightpanel shows the antibody recruiting capability of 10 uM ARM-U2 6-ABT-1represented again as a dot plot with the gates assigned as described for1-ABT-1. Note the significantly higher percentage of antibody binding tothe cell surface of 6-ABT-1 vs 1-ABT-1 (left panel) depicted as a shiftin FL-4 fluorescence with almost all the cells present in the lowerright gate. Below. histogram compares the antibody recruiting capabilityof derivatives 1-ABT-1 with its longer linker counterparts 1-ABT-2 and1-ABT-3 all at 10 uM concentrations highlighting lower shift in FL-4fluorescence corresponding to the decreased potency of antibodyrecruitment accompanying increasing linker length.

FIG. 16 shows the uPAR-dependant simultaneous binding of 6-ABT-1 to boththe surface of uPAR+ glioblastoma A172 cells and anti-DNP antibodiesevaluated using a flow cytometry-based antibody-recruiting in vitroassay. Binding was detected by the addition of anti-DNP antibody plusstreptavidin-AlexaFluor647 following the addition of 10 uM or 1 uM6-ABT-1 (upper left and lower left panels respectively), and could beoutcompeted upon the addition of 2.1 uM soluble recombinant human uPARdisplacing 6-ABT-1 off the cell surface (upper right panel-10 uM 6-ABT-1displaced, lower right panel-1 uM 6-ABT-1 displaced).

FIG. 17 shows that compound 6-ABT-1 selectively binds to uPAR andstimulates immune responses against uPAR+ cancer targets. Left. 10M of6-ABT-1 minimally bound to uPAR− negative B16 vector-transfectedmelanoma cells. Right. 10 μM of 6-ABT-1 bound strongly to B16 melanomacells transfected with human uPAR Compound binding was detected bybiotinylated anti-DNP antibody plus streptavidin-AlexaFluor647.

FIG. 18 shows phagocytosis of A172 target cells by U937 monocytic cellsinduced by shortest linker ABT-1 series ARM-U2 derivatives in thepresence of anti-DNP antibodies illustrating the increased efficacy andpotency of double sulfonate derivative 6-ABT-1.

FIG. 19 shows a dot-plot representation of 6-ABT-1 concentration screen(A-F) and out-competition selectivity experiments (I-VI) carried outusing a flow cytometry-based dual fluorescence ADCP assay. Upper rightrectangular gates represent dual positive cell events indicative ofphagocytosis, Lower right rectangular gates represent target A172 cellonly cell events, Ungated effector cell population present on upperright corner of dot plot. A. 10 nM 6-ABT-1 B. 20 nM 6-ABT-1 C. 50 nM6-ABT-1 D. 100 nM 6-ABT-1 E. 1 uM 6-ABT-1 F. 10 uM 6-ABT-1 I. 20 nM6-ABT-1 II. 20 nM 6-ABT-1+20 uM 6-ABT-4 III. 20 nM 6-ABT-1+2.1 uM uPARIV. 50 nM 6-ABT-1 V. 50 nM 6-ABT-1+20 uM 6-ABT-4 VI. 50 nM 6-ABT-1+2.1uM uPAR

FIG. 20 shows A. Phagocytosis of A172 target cells by U937 monocyticcells induced by parent mono acid derivatives 1-ABT-1/2/3 illustratingthe decreased potency associated with increasing antibody bindingterminus liker length. B. Identical assay as in A. above only withdisulfonate derivatives 6-ABT-1/2/3 illustrating a significant increasein efficacy and potency relative to parent derivatives 1-ABT-1/2/3 inpanel A in addition to a modest decrease in potency with increasinglinker length beyond a PEG-8 spacer.

FIG. 21 shows Flow cytometry ADCP assay analysis of B16 target cellstransfected with a human uPAR cloned plasmid vs transfected with anempty vector, by U937 monocytic cells induced by derivative 6-ABT-1,demonstrating the specificity of 6-ABT-1 for cell surface uPAR.

FIG. 22 shows histogram representation of a multi-inflammatory ELISAexperiment used to assay the ability of ARM-U2 6-ABT-1 to induce u937effector cytokine release in the presence of A172 target cells.Indicated is a significant enhancement in the release of IL-8 by u937cells induced by 6-ABT in the presence of target cells plus anti-DNPantibodies

FIG. 23 shows the evaluation of binding affinity of ARM-U2 degradationproduct to 23 using a competitive ELISA. An IC₅₀ of 63 nM was observedtranslating to calculated K_(I) of 10 nM demonstrating no change in uPARbinding affinity relative to ARM-U2.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention there may be employedconventional chemical synthetic and pharmaceutical formulation methods,as well as pharmacology, molecular biology, microbiology, andrecombinant DNA techniques within the skill of the art. Such techniquesare well-known and are otherwise explained fully in the literature.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise (such as in the case of a groupcontaining a number of carbon atoms), between the upper and lower limitof that range and any other stated or intervening value in that statedrange is encompassed within the invention. The upper and lower limits ofthese smaller ranges may independently be included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

It is to be noted that as used herein and in the appended claims, thesingular forms “a,” “an”, “and” and “the” include plural referencesunless the context clearly dictates otherwise.

Furthermore, the following terms shall have the definitions set outbelow. It is understood that in the event a specific term is not definedhereinbelow, that term shall have a meaning within its typical usewithin context by those of ordinary skill in the art.

The term “compound”, as used herein, unless otherwise indicated, refersto any specific chemical compound disclosed herein and includestautomers, regioisomers, geometric isomers, stereoisomers and whereapplicable, optical isomers (enantiomers) thereof, as well aspharmaceutically acceptable salts and derivatives (including prodrugforms) thereof. Within its use in context, the term compound generallyrefers to a single compound, but also may include other compounds suchas stereoisomers, regioisomers and/or optical isomers (including racemicmixtures) as well as specific enantiomers or enantiomerically enrichedmixtures of disclosed compounds. The term also refers, within context,to prodrug forms of compounds which have been modified to facilitate theadministration and delivery of compounds to a site of activity. It isnoted that in describing the present compounds, numerous substituents,linkers and connector molecules and variables associated with same,among others, are described. The use of a bond presented as -----signifies that a single bond is present or absent, depending on thecontext of the chemistry described. The use of a bond presented as

signifies that a single bond or a double bond is intended depending onthe context of the chemistry described. It is understood by those ofordinary skill that molecules which are described herein are stablecompounds as generally described hereunder. Active compounds accordingto the present invention which bind to the urokinase-type plasminogenactivator receptor are collectively referred to as ARM-U2 compounds, aswell as difunctional compounds (even where the compounds aremultifunctional).

The term “patient” or “subject” is used throughout the specificationwithin context to describe an animal, generally a mammal and preferablya human, to whom treatment, including prophylactic treatment(prophylaxis, including especially as that term is used with respect toreducing the likelihood of metastasis of an existing cancer), with thecompositions according to the present invention is provided. Fortreatment of those infections, conditions or disease states which arespecific for a specific animal such as a human patient or a patient of aparticular gender, such as a human male or female patient, the termpatient refers to that specific animal. Compounds according to thepresent invention are useful for the treatment of cancer, includingespecially for use in reducing the likelihood of metastasis of a cancer.

The term “effective” is used herein, unless otherwise indicated, todescribe an amount of a compound or composition which, in context, isused to produce or effect an intended result, whether that resultrelates to the inhibition of the effects of a disease state (e.g.cancer) on a subject or the treatment or prophylaxis of a subject forsecondary conditions, disease states or manifestations of disease statesas otherwise described herein. This term subsumes all other effectiveamount or effective concentration terms (including the term“therapeutically effective”) which are otherwise described in thepresent application.

The terms “treat”, “treating”, and “treatment”, etc., as used herein,refer to any action providing a benefit to a patient at risk for canceror metastasis of cancer, including improvement in the condition throughlessening or suppression of at least one symptom, inhibition of cancergrowth, reduction in cancer cells or tissue, prevention, reduction inthe likelihood or delay in progression of cancer or metastasis of thecancer, prevention or delay in the onset of disease states or conditionswhich occur secondary to cancer or remission or cure of the cancer,among others. Treatment, as used herein, encompasses both prophylacticand therapeutic treatment. The term “prophylactic” when used, means toreduce the likelihood of an occurrence or the severity of an occurrencewithin the context of the treatment of cancer, including cancermetastasis as otherwise described hereinabove.

The term “neoplasia” or “cancer” is used throughout the specification torefer to the pathological process that results in the formation andgrowth of a cancerous or malignant neoplasm, i.e., abnormal tissue thatgrows by cellular proliferation, often more rapidly than normal andcontinues to grow after the stimuli that initiated the new growth cease.Malignant neoplasms show partial or complete lack of structuralorganization and functional coordination with the normal tissue and mostinvade surrounding tissues, metastasize to several sites, and are likelyto recur after attempted removal and to cause the death of the patientunless adequately treated. As used herein, the term neoplasia is used todescribe all cancerous disease states and embraces or encompasses thepathological process associated with malignant hematogenous, ascitic andsolid tumors.

Neoplasms include, without limitation, morphological irregularities incells in tissue of a subject or host, as well as pathologicproliferation of cells in tissue of a subject, as compared with normalproliferation in the same type of tissue. Additionally, neoplasmsinclude benign tumors and malignant tumors (e.g., colon tumors) that areeither invasive or noninvasive. Malignant neoplasms (cancer) aredistinguished from benign neoplasms in that the former show a greaterdegree of anaplasia, or loss of differentiation and orientation ofcells, and have the properties of invasion and metastasis. Examples ofneoplasms or neoplasias from which the target cell of the presentinvention may be derived include, without limitation, carcinomas (e.g.,squamous-cell carcinomas, adenocarcinomas, hepatocellular carcinomas,and renal cell carcinomas), particularly those of the bladder, bowel,breast, cervix, colon, esophagus, head, kidney, liver, lung, neck,ovary, pancreas, prostate, and stomach; leukemias; benign and malignantlymphomas, particularly Burkitt's lymphoma and Non-Hodgkin's lymphoma;benign and malignant melanomas; myeloproliferative diseases; sarcomas,particularly Ewing's sarcoma, hemangiosarcoma, Kaposi's sarcoma,liposarcoma, myosarcomas, peripheral neuroepithelioma, and synovialsarcoma; tumors of the central nervous system (e.g., gliomas,astrocytomas, oligodendrogliomas, ependymomas, gliobastomas,neuroblastomas, ganglioneuromas, gangliogliomas, medulloblastomas,pineal cell tumors, meningiomas, meningeal sarcomas, neurofibromas, andSchwannomas); germ-line tumors (e.g., bowel cancer, breast cancer,prostate cancer, cervical cancer, uterine cancer, lung cancer, ovariancancer, testicular cancer, thyroid cancer, astrocytoma, esophagealcancer, pancreatic cancer, stomach cancer, liver cancer, colon cancer,and melanoma); mixed types of neoplasias, particularly carcinosarcomaand Hodgkin's disease; and tumors of mixed origin, such as Wilms' tumorand teratocarcinomas (Beers and Berkow (eds.), The Merck Manual ofDiagnosis and Therapy, 17.sup.th ed. (Whitehouse Station, N.J.: MerckResearch Laboratories, 1999) 973-74, 976, 986, 988, 991). All of theseneoplasms may be treated using compounds according to the presentinvention.

Representative common cancers to be treated with compounds according tothe present invention include, for example, prostate cancer, metastaticprostate cancer, stomach, colon, rectal, liver, pancreatic, lung,breast, cervix uteri, corpus uteri, ovary, testis, bladder, renal,brain/CNS, head and neck, throat, Hodgkin's disease, non-Hodgkin'slymphoma, multiple myeloma, leukemia, melanoma, non-melanoma skincancer, acute lymphocytic leukemia, acute myelogenous leukemia, Ewing'ssarcoma, small cell lung cancer, choriocarcinoma, rhabdomyosarcoma,Wilms' tumor, neuroblastoma, hairy cell leukemia, mouth/pharynx,oesophagus, larynx, kidney cancer and lymphoma, among others, which maybe treated by one or more compounds according to the present invention.Because of the activity of the present compounds, the present inventionhas general applicability treating virtually any cancer in any tissue,thus the compounds, compositions and methods of the present inventionare generally applicable to the treatment of cancer and in reducing thelikelihood of development of cancer and/or the metastasis of an existingcancer.

In certain particular aspects of the present invention, the cancer whichis treated is metastatic cancer, a recurrent cancer or a drug resistantcancer, especially including a drug resistant cancer. Separately,metastatic cancer may be found in virtually all tissues of a cancerpatient in late stages of the disease, typically metastatic cancer isfound in lymph system/nodes (lymphoma), in bones, in lungs, in bladdertissue, in kidney tissue, liver tissue and in virtually any tissue,including brain (brain cancer/tumor). Thus, the present invention isgenerally applicable and may be used to treat any cancer in any tissue,regardless of etiology.

The term “tumor” is used to describe a malignant or benign growth ortumefacent.

The term “antibody binding moiety”, “antibody binding terminus” or“antibody binding-structure” (A_(B)M or ABT, which abbreviations areused synonymously) within the general formula of compounds according tothe present invention) is used to described that portion of abifunctional ARM-U2 compound according to the present invention whichcomprises at least one small molecule or hapten which can bind toantibodies within the patient. The term “hapten” is used to describe asmall-molecular-weight inorganic or organic molecule that alone is notantigenic but which when linked to another molecule, such as a carrierprotein (albumin, etc.) or in the case of the present invention, as anantibody terminus in the present compounds, is antigenic; and anantibody raised against the hapten (generally, the hapten bonded orcomplexed to the carrier) will react with the hapten alone. Because, inmany instances, anti-hapten (especially anti-DNP) antibodies are alreadypresent in the human blood stream as endogenous antibodies because theynaturally become raised to endogenous haptens (already present inpatients), no pre-vaccination is necessary for ARM-U2 activity, but mayoptionally be used to increase the efficacy of the ARM-U2 compoundsdisclosed herein.

It is preferred that the antibody binding moiety comprise a hapten whichis reactive with (binds to) an endogenous antibody that pre-exists inthe patient prior to initiation of therapy with the compounds of thepresent invention and does not have to be separately raised as part of atreatment regimen (for example, by vaccination or other approach forenhancing immunogenicity), which is optionally used in the presentinvention. Thus, haptens which comprise a di- or trinitro phenyl groupor a rhamnose group, or a digalactose hapten (Gal-Gal-Z, preferablyGal-Gal-sugar, preferably Gal-Gal-Glu), are preferred. Additionally, acompound according to the general structure:

-   Where X″ is O, CH₂, NR¹, S; and-   R¹ is H, a C₁-C₃ alkyl group or a —C(O)(C₁-C₃) group;-   May be used as haptens in the present invention.

Further, a moiety according to the chemical structure:

-   Where X^(b) is a bond, O, CH₂, NR¹ (as above) or S may also be used    as a hapten (A_(B)M) in the present invention.

A preferred A_(B)M moiety is:

Additional A_(B)M moieties include the following:

-   Where R^(NO2) is a nitrophenyl group or a dinitrophenyl group which    is bonded to the adjacent amine group or thio group as indicated;

-   a group according to the chemical structure:

-   Where Y′ is H or NO₂ (preferably H);-   X is O, CH₂, NR¹, S, S(O), S(O)₂, —S(O)₂O, —OS(O)₂, or OS(O)₂O; and-   R¹ is H, a C₁-C₃ alkyl group, or a —C(O)(C₁-C₃) group;

The fluorescein hapten (A_(B)M) moiety for use in the present inventionis represented by the chemical structure (as a racemic mixture, or aseither enantiomer) and may also be used as a hapten for use in thepresent invention:

The (Gal-Gal-Z) hapten is represented by the chemical formula:

-   Where X′ is CH₂, O, N—R^(1′), or S, preferably O;-   R^(1′) is H or C₁-C₃ alkyl; and-   Z is a bond, a monosaccharide, disaccharide, oligosaccharide,    glycoprotein or glycolipid, preferably a sugar group, more    preferably a sugar group selected from the monosaccharides,    including aldoses and ketoses, and disaccharides, including those    disaccharides described herein. Monosaccharide aldoses include    monosaccharides such as aldotriose (D-glyceraldehdye, among others),    aldotetroses (D-erythrose and D-Threose, among others),    aldopentoses, (D-ribose, D-arabinose, D-xylose, D-lyxose, among    others), aldohexoses (D-allose, D-altrose, D-Glucose, D-Mannose,    D-gulose, D-idose, D-galactose and D-Talose, among others), and the    monosaccharide ketoses include monosaccharides such as ketotriose    (dihydroxyacetone, among others), ketotetrose (D-erythrulose, among    others), ketopentose (D-ribulose and D-xylulose, among others),    ketohexoses (D-Psicone, D-Fructose, D-Sorbose, D-Tagatose, among    others), aminosugars, including galactoseamine, sialic acid,    N-acetylglucosamine, among others and sulfosugars, including    sulfoquinovose, among others. Exemplary disaccharides which find use    in the present invention include sucrose (which may have the glucose    optionally N-acetylated), lactose (which may have the galactose    and/or the glucose optionally N-acetylated), maltose (which may have    one or both of the glucose residues optionally N-acetylated),    trehalose (which may have one or both of the glucose residues    optionally N-acetylated), cellobiose (which may have one or both of    the glucose residues optionally N-acetylated), kojibiose (which may    have one or both of the glucose residues optionally N-acetylated),    nigerose (which may have one or both of the glucose residues    optionally N-acetylated), isomaltose (which may have one or both of    the glucose residues optionally N-acetylated), β,β-trehalose (which    may have one or both of the glucose residues optionally    N-acetylated), sophorose (which may have one or both of the glucose    residues optionally N-acetylated), laminaribiose (which may have one    or both of the glucose residues optionally N-acetylated),    gentiobiose (which may have one or both of the glucose residues    optionally N-acetylated), turanose (which may have the glucose    residue optionally N-acetylated), maltulose (which may have the    glucose residue optionally N-acetylated), palatinose (which may have    the glucose residue optionally N-acetylated), gentiobiluose (which    may have the glucose residue optionally N-acetylated), mannobiose,    melibiose (which may have the glucose residue and/or the galactose    residue optionally N-acetylated), melibiulose (which may have the    galactose residue optionally N-acetylated), rutinose, (which may    have the glucose residue optionally N-acetylated), rutinulose and    xylobiose, among others. Oligosaccharides for use in the present    invention as Z can include any sugar of three or more (up to    about 100) individual sugar (saccharide) units as described above    (i.e., any one or more saccharide units described above, in any    order, especially including glucose and/or galactose units as set    forth above), or for example, fructo-oligosaccharides,    galactooligosaccharides and mannan-oligosaccharides ranging from    three to about ten-fifteen sugar units in size. Glycoproteins for    use in the present invention include, for example, N-glycosylated    and O-glycosylated glycoproteins, including the mucins, collagens,    transferrin, ceruloplasmin, major histocompatability complex    proteins (MHC), enzymes, lectins and selectins, calnexin,    calreticulin, and integrin glycoprotein IIb/IIa, among others.    Glycolipids for use in the present invention include, for example,    glyceroglycolipids (galactolipids, sulfolipids), glycosphingolipids,    such as cerebrosides, galactocerebrosides, glucocerebrosides    (including glucobicaranateoets), gangliosides, globosides,    sulfatides, glycophosphphingolipids and glycocalyx, among others.

Preferably, Z is a bond (linking a Gal-Gal disaccharide to a linker orconnector molecule) or a glucose or glucosamine (especiallyN-acetylglucosamine).

It is noted that Z is linked to a galactose residue through a hydroxylgroup or an amine group on the galactose of Gal-Gal, preferably ahydroxyl group. A preferred hapten is Gal-Gal-Glu which is representedby the structure:

-   Where Xs is OH or NHAc.-   Other A_(B)M groups include, for example, the following groups:

-   Where X_(R) is O, S or NR¹; and-   X_(M) is O, NR¹ or S, and-   R¹ is H, a C₁-C₃ alkyl group or a —C(O)(C₁-C₃) group, or a    pharmaceutically acceptable salt form or alternative salt form    thereof.

Noted is that more than one rhamnose group (preferably from 1 to 4rhamnose groups) may be used in the present compounds to provideenhanced antibody recruitment activity.

It is noted in the carboxyethyl lysine A_(B)M moiety either one, two orthree of the nitrogen groups may be linked to the remaining portion ofthe molecule through the linker or one or both of the remaining nitrogengroups may be substituted with a dinitrophenyl through an X group asotherwise described herein.

The term “urokinase-type plasminogen activator receptor”, “uPAR” orsimply “urokinase receptor” is used to describe a receptor which is theactive site for binding of the present compounds on cancer cellspursuant to the present invention. These receptors are oftenoverexpressed in cancer cells are are known to promote invasion,migration, and metastasis in cancer cells. Accordingly, the presentARM-U2 compounds exhibit two distinguishable inhibitory actions oncancer cells. The first is to inhibit the activity of uPAR by binding tothe receptor resulting in the cancer cells to which the presentcompounds bind having their ability to invade, migrate and metastasizeinhibited. The second is to function as antibody recruitment compoundswhich recruit antibodies to cancer cells selectively once bound to uPAR,resulting in an antibody response to further inhibit, cause cell deathand otherwise treat the cancer cells in the patient administeredcompounds according to the invention. uPAR is a part of the plasminogenactivation system, which in a healthy, non-cancerous body is involved intissue reorganization events such as mammary gland involution and woundhealing. In order to be able to reorganize tissue, the old tissue mustbe able to be degraded. An important mechanism in this degradation isthe proteolysis cascade initiated by the plasminogen activation system.uPAR binds urokinase and thus restricts plasminogen activation to theimmediate vicinity of the cell membrane. uPAR is believed to play animportant role in the regulation of this process. However, thecomponents of the plasminogen activation system have been found to behighly expressed in many malignant tumors, indicating that tumors areable to hijack the system, and use it in metastatis. Accordingly,compounds of the present invention, at least in part, act as inhibitorsof the plasminogen activation system as potent anticancer agents.

The term “urokinase-type plasminogen activator receptor binding moiety”or “UPAR_(B)M” is a moiety which binds to an active site ofurokinase-type plasminogen activator receptor (uPAR) on the surface ofcancer cells of a patient or subject and is used to described thatportion of an ARM-U2 compound according to the present invention whichcomprises at least one small molecule or moiety which can bind tourokinase-type plasminogen activator receptor and can be used to produceARM-U2 compounds hereof. The binding which occurs is competitive andmaintains the compound in uPAR in order to inhibit the ability of thecancer cells to invade, migrate and/or metastasize and separately, toattract antibodies to those same cancer cells.

Preferred UPAR_(B)M groups for use in the present invention are setforth below. In one embodiment, the UPAR_(B)M group is a moiety (or itsenantiomer with the acyl group of the amide being disposed upwardsrather than downward from the plane as depicted) according to thechemical structure:

-   where R₁, R₂, R₃, R₄ and R₅ are each independently H, a halogen (F,    Cl, Br, I, preferably F), a C₁-C₃ alkyl group optionally substituted    with one or two hydroxyl groups or up to three fluoro groups, a    (CH₂)_(m′)COOH group, a (CH₂)_(m′)SO₃H group,-   or

-   Each R′ is independently H or a C₁-C₃ alkyl group (preferably H or    CH₃, most often H);-   m′ is 0, 1, 2 or 3 (preferably 0 in the case of the sulfonic acid or    1 in the case of the carboxylic acid); and-   y′ is 0, 1 or 2 (preferably 0 or 1.

Alternatively, preferred UPAR_(B)M groups ((or its enantiomer with theacyl group of the amide being disposed upwards rather than downward fromthe plane as depicted)) are directed to the following groups:

-   Wherein each R^(N) is independently H or a C₁-C₃ alkyl group;-   R₁, R₂, R₃, R₄ and R₅ are each independently H, a halogen (F, Cl,    Br, I, preferably F),-   a C₁-C₃ alkyl group optionally substituted with one or two hydroxyl    groups or up to three fluoro groups, a (CH₂)_(m′)COOH group, a    (CH₂)_(m′)SO₃H group,-   or

-   Each R′ is independently H or a C₁-C₃ alkyl group (preferably H or    CH₃, most often H);-   m′ is 0, 1, 2 or 3 (preferably 0 in the case of the sulfonic acid or    1 in the case of the carboxylic acid); and-   y′ is 0, 1 or 2 (preferably 0 or 1.

The UPAR_(B)M groups are attached through the indicated amine group to aR^(LABT) group.

The R^(LABT) group is represented as an group,

-   where L is a bond, at least one linker (preferably a single linker)    which comprises a first linker group L1 which optionally includes a    connector group CT and an optional linker group L2 which itself    optionally includes a connector group CT, said first linker group L1    being linked to said second linker group L2 optionally (preferably)    through a CT group; and-   is an antibody bonding moiety comprising a hapten which is capable    of binding to an antibody in said patient or subject as otherwise    described herein. The ARM-U2 compounds according to the present    invention may be a pharmaceutically acceptable salt, solvate or    polymorph thereof.

In preferred embodiments of the invention, R₁ is H or SO₃H, R₂ is H,CO₂H, SO₃H, —NHCH₂—CO₂H or —C(O)—NHCH₂—CO₂H, R₃ is H or SO₃H and R₄ is Hor CO₂H, or a pharmaceutically acceptable salt or solvate thereof.

The term “pharmaceutically acceptable salt” is used throughout thespecification to describe a salt form of one or more of the compoundsherein which are presented to increase the solubility of the compound insaline for parenteral delivery or in the gastric juices of the patient'sgastrointestinal tract in order to promote dissolution and thebioavailability of the compounds. Pharmaceutically acceptable saltsinclude those derived from pharmaceutically acceptable inorganic ororganic bases and acids. Suitable salts include those derived fromalkali metals such as potassium and sodium, alkaline earth metals suchas calcium, magnesium and ammonium salts, among numerous other acidswell known in the pharmaceutical art. Sodium and potassium salts may beparticularly preferred as neutralization salts of carboxylic acidcontaining compositions according to the present invention. The term“salt” shall mean any salt consistent with the use of the compoundsaccording to the present invention. In the case where the compounds areused in pharmaceutical indications, including the treatment of HIVinfections, the term “salt” shall mean a pharmaceutically acceptablesalt, consistent with the use of the compounds as pharmaceutical agents.

The term “linker”, “L1” or “L2” refers to a chemical entity connectingan antibody binding (A_(B)M) moiety to a urokinase-like plasminogenactivator receptor binding moiety (UPAR_(B)M), optionally through atleast one (preferably one) connector moiety (CT) through covalent bonds.The linker between the two active portions of the molecule, that is theantibody binding moiety (A_(B)M) and the urokinase binding moiety(UPAR_(B)M) ranges from about 5 Å to about 50 Å or more in length, about6 Å to about 45 Å in length, about 7 Å to about 40 Å in length, about 8Å to about 35 Å in length, about 9 Å to about 30 Å in length, about 10 Åto about 25 Å in length, about 7 Å to about 20 Å in length, about 5 Å toabout 16 Å in length, about 5 Å to about 15 Å in length, about 6 Å toabout 14 Å in length, about 10 Å to about 20 Å in length, about 11 Å toabout 25 Å in length, etc. Linkers which are based upon ethylene glycolunits and are between 2 and 15 glycol units, 1 and 8 glycol units, 1, 2,3, 4, 5, and 6 glycol units in length may be preferred. By having alinker with a length as otherwise disclosed herein, the A_(B)M moietyand the UPAR_(B)M moiety may be situated to advantageously takeadvantage of the biological activity of compounds according to thepresent invention which bind to urokinase-like plasminogen activator oncancer cells, including cancer cells prone to metastasis and attractendogenous antibodies to those cells to which the compounds are bound,resulting in the selective and targeted death of those cells. Theselection of a linker component is based on its documented properties ofbiocompatibility, solubility in aqueous and organic media, and lowimmunogenicity/antigenicity. Although numerous linkers may be used asotherwise described herein, a linker based upon polyethyleneglycol (PEG)linkages, polypropylene glycol linkages, orpolyethyleneglycol-co-polypropylene oligomers (up to about 100 units,about 1 to 100, about 1 to 75, about 1 to 60, about 1 to 50, about 1 to35, about 1 to 25, about 1 to 20, about 1 to 15, 2 to 10, about 4 to 12,about 1 to 8, 1 to 3, 1 to 4, 2 to 6, 1 to 5, etc.) may be favored as alinker because of the chemical and biological characteristics of thesemolecules. The use of polyethylene (PEG) linkages is preferred. Whendescribing linkers according to the present invention, includingpolyethylene glycol linkers or other linkers, one or more additionalgroups (e.g., methylene groups, amide groups, etc., methylene groups arepreferred) may be covalently attached at either end of the linker groupto attach to a UPAR_(B)M group, a CT group, another linker group or anA_(B)M group.

Alternative linkers may include, for example, polyamino acid linkers ofup to 100 amino acids (of any type, preferably D- or L-amino acids,preferably naturally occurring L-amino acids) in length (m is about 1 to100, about 1 to 75, about 1 to 60, about 1 to 50, about 1 to 45, about 1to 35, about 1 to 25, about 1 to 20, about 1 to 15, 2 to 10, about 4 to12, about 5 to 10, about 4 to 6, about 1 to 8, about 1 to 6, about 1 to5, about 1 to 4, about 1 to 3, etc.), optionally including one or twoconnecting groups (preferably at one or both ends of the polyamino acidlinker).

Preferred linkers include those according to the chemical structures:

-   Or a polypropylene glycol or polypropylene-co-polyethylene glycol    linker having between 1 and 100 alkylene glycol units;-   Where R_(a) is H, C₁-C₃ alkyl or alkanol or forms a cyclic ring with    R³ (proline) and R³ is a side chain derived from a D- or L amino    acid (preferably a naturally occurring L-amino acid) preferably    selected from the group consisting of alanine (methyl), arginine    (propyleneguanidine), asparagine (methylenecarboxyamide), aspartic    acid (ethanoic acid), cysteine (thiol, reduced or oxidized    di-thiol), glutamine (ethylcarboxyamide), glutamic acid (propanoic    acid), glycine (H), histidine (methyleneimidazole), isoleucine    (1-methylpropane), leucine (2-methylpropane), lysine    (butyleneamine), methionine (ethylmethylthioether), phenylalanine    (benzyl), proline (R³ forms a cyclic ring with R_(a) and the    adjacent nitrogen group to form a pyrrolidine group),    hydroxyproline, serine (methanol), threonine (ethanol,    1-hydroxyethane), tryptophan (methyleneindole), tyrosine (methylene    phenol) or valine (isopropyl);-   m (within the context of this use) is an integer from 1 to 100, 1 to    75, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to 40, 2 to 35, 3 to 30, 1    to 15, 1 to 10, 1 to 8, 1 to 6, 1, 2, 3, 4 or 5;-   n (within the context of this use) is an integer from about 1 to    100, about 1 to 75, about 1 to 60, about 1 to 50, about 1 to 45,    about 1 to 35, about 1 to 25, about 1 to 20, about 1 to 15, 2 to 10,    about 4 to 12, about 5 to 10, about 4 to 6, about 1 to 8, about 1 to    6, about 1 to 5, about 1 to 4, about 1 to 3, etc.) or

Another linker according to the present invention comprises apolyethylene glycol linker containing from 1 to 1 to 100, 1 to 75, 1 to60, 1 to 55, 1 to 50, 1 to 45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to10, 1 to 8, 1 to 6, 1, 2, 3, 4 or 5 ethylene glycol units, to which isbonded a lysine group (preferably at its carboxylic acid moiety) whichbinds one or two DNP groups to the lysine at the amino group(s) oflysine. Still other linkers comprise amino acid residues (D or L) towhich are bonded to A_(B)M moieties, in particular, DNP, among others atvarious places on amino acid residue as otherwise described herein. Inanother embodiment, as otherwise described herein, the amino acid hasanywhere from 1-15 methylene groups separating the amino group from theacid group in providing a linker to the A_(B)M moiety.

Or another linker is according to the chemical formula:

-   Where Z and Z′ are each independently a bond, —(CH₂)_(i)—O,    —(CH₂)_(i)—S, —(CH₂)_(i)—N—R,

wherein said —(CH₂)_(i) group, if present in Z or Z′, is bonded to aconnector (CT), A_(B)M and/or U_(k)BM;

-   Each R is H, or a C₁-C₃ alkyl or alkanol group;-   Each R² is independently H or a C₁-C₃ alkyl group;-   Each Y is independently a bond, O, S or N—R;-   Each i is independently 0 to 100, 0 to 75, 1 to 60, 1 to 55, 1 to    50, 1 to 45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1    to 6, 0, 1, 2, 3, 4 or 5;-   D is

-   a bond, with the proviso that Z, Z′ and D are not each    simultaneously bonds;-   j is 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to 40,    2 to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1, 2, 3, 4 or 5;-   m′ is 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to    40, 2 to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1, 2, 3, 4    or 5;-   n is 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to 40,    2 to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1, 2, 3, 4 or 5    (n is preferably 2);-   X¹ is O, S or N—R; and-   R is as described above, or a pharmaceutical salt thereof.

Other linkers which are included herein include preferred linkersaccording to the chemical structure:

-   where each n and n′ is independently 1 to 25, 1 to 15, 1 to 12, 2 to    11, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4 and 2 to 3 or 1, 2, 3,    4, 5, 6, 7, or 8; and-   each n″ is independently 0 to 8, often 1 to 7, or 1, 2, 3, 4, 5 or 6    (preferably 3).

Preferred linkers which include a CT group (especially a diamide CTgroup as otherwise described herein) connecting a first and second (PEG)linker group include the following structures:

-   where each n and n′ is independently 1 to 25, 1 to 15, 1 to 12, 2 to    11, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4 and 2 to 3 or 1, 2, 3,    4, 5, 6, 7, or 8; and-   each n″ is independently 0 to 8, often 1 to 7, or 1, 2, 3, 4, 5 or 6    (preferably 3). Noted is that each of these linkers may contain    alkylene groups containing from 1 to 4 methylene groups at the    distal ends of each linker group in order to facilitate connection    of the linker group.

The term “connector”, symbolized in the generic formulas by (CT), isused to describe a chemical moiety which is optionally included inbifunctional compounds according to the present invention which formsfrom the reaction product of an activated A_(B)M-linker with a U_(k)BMmoiety (which also is preferably activated) or an A_(B)M moiety with anactivated linker-U_(k)BM as otherwise described herein. The connectorgroup is often the resulting moiety which forms from the facilecondensation of two or more separate chemical fragments which containreactive groups which can provide connector groups as otherwisedescribed to produce bifunctional or multifunctional compounds accordingto the present invention. It is noted that a connector may bedistinguishable from a linker in that the connector is the result of aspecific chemistry which is used to provide bifunctional compoundsaccording to the present invention wherein the reaction product of thesegroups results in an identifiable connector group or part of a connectorgroup which is distinguishable from the linker group, although incertain instances, the connector group is incorporated into and integralwith the linker group as otherwise described herein. It is noted alsothat a connector group may be linked to a number of linkers to providemultifunctionality (i.e., more than one U_(k)BM moiety and/or more thanone A_(B)M moiety within the same molecule. It is noted that there maybe some overlap between the description of the connector group and thelinker group such that the connector group is actually incorporated orforms part of the linker, especially with respect to more commonconnector groups such as amide groups, oxygen (ether), sulfur(thioether) or amine linkages, urea or carbonate —OC(O)O— groups asotherwise described herein. It is further noted that a connector (orlinker) may be connected to A_(B)M, a linker or U_(k)BM at positionswhich are represented as being linked to another group using the symbol

Where two or more such groups are present in a linker or connector, anyof an A_(B)M, a linker or a U_(k)BM may be bonded to such a group. Wherethat symbol is not used, the linker may be at one or more positions of amoiety.

Common connector groups which are used in the present invention includethe following chemical groups:

-   -   or a diamide group according to the structure:

-   Where X² is CH₂, O, S, NR⁴, C(O), S(O), S(O)₂, —S(O)₂O, —OS(O)₂, or    OS(O)₂O;-   X³ is O, S, NR⁴;-   R⁴ is H, a C₁-C₃ alkyl or alkanol group, or a —C(O)(C₁-C₃) group;-   R¹ is H or a C₁-C₃ alkyl group (preferably H); and-   n″ is independently 0 to 8, often 1 to 7, or 1, 2, 3, 4, 5 or 6    (preferably 3). The triazole group, indicated above, is a preferred    connector group. It is noted that each connector may be extended    with one or more methylene groups to facilitate connection to a    linker group, another CT group, a UPAR_(B)M group or a A_(B)M group.    It is noted that in certain instances, within context the diamide    group may also function independently as a linker group.

It is noted that each of the above groups may be further linked to achemical moiety which bonds two or more of the above connector groupsinto a multifunctional connector, thus providing complex multifunctionalcompounds comprising more than one A_(B)M and/or UPAR_(B)M group and anumber of linker groups within the multifunctional compound.

The term “alkyl” refers to a fully saturated monovalent radicalcontaining carbon and hydrogen, and which may be cyclic, branched or astraight chain containing from 1 to 10 carbon atoms, (1, 2, 3, 4, 5, 6,7, 8, 9 or 10), preferably 1, 2 or 3 carbon atoms. Examples of alkylgroups are methyl, ethyl, n-butyl, n-hexyl, n-heptyl, n-octyl,isopropyl, 2-methylpropyl, cyclopropyl, cyclopropylmethyl, cyclobutyl,cyclopentyl, cyclopen-tylethyl, cyclohexylethyl and cyclohexyl.Preferred alkyl groups are C₁-C₆ or C₁-C₃ alkyl groups. “Alkylene”(e.g., methylene) when used, refers to a fully saturated hydrocarbonwhich is divalent (may be linear, branched or cyclic) and which isoptionally substituted. Other terms used to indicate substitutent groupsin compounds according to the present invention are as conventionallyused in the art.

The term “coadministration” shall mean that at least two compounds orcompositions are administered to the patient at the same time, such thateffective amounts or concentrations of each of the two or more compoundsmay be found in the patient at a given point in time. Although compoundsaccording to the present invention may be co-administered to a patientat the same time, the term embraces both administration of two or moreagents at the same time or at different times, provided that effectiveconcentrations of all coadministered compounds or compositions are foundin the subject at a given time. ARM-U2 compounds according to thepresent invention may be administered with one or more additionalanti-cancer agents or other agents which are used to treat or amelioratethe symptoms of cancer, especially including metastatic cancer.Exemplary anticancer agents which may be coadministered in combinationwith one or more chimeric compounds according to the present inventioninclude, for example, antimetabolites, inhibitors of topoisomerase Iand/or II, alkylating agents and microtubule inhibitors (e.g., taxol),among numerous others, as otherwise described herein.

The term “additional anti-cancer agent” refers to one or moretraditional cancer agent(s) which may be co-administered with compoundsaccording to the present invention in the treatment of cancer. Theseagents include chemotherapeutic agents and include one or more membersselected from the group consisting of everolimus, trabectedin, abraxane,TLK 286, AV-299, DN-101, pazopanib, GSK690693, RTA 744, ON 0910.Na, AZD6244 (ARRY-142886), AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin,vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263, aFLT-3 inhibitor, a VEGFR inhibitor, an EGFR TK inhibitor, an aurorakinase inhibitor, a PIK-1 modulator, a Bcl-2 inhibitor, an HDACinhibitor, a c-MET inhibitor, a PARP inhibitor, a Cdk inhibitor, an EGFRTK inhibitor, an IGFR-TK inhibitor, an anti-HGF antibody, a PI3 kinaseinhibitors, an AKT inhibitor, a JAK/STAT inhibitor, a checkpoint-1 or 2inhibitor, a focal adhesion kinase inhibitor, a Map kinase kinase (mek)inhibitor, a VEGF trap antibody, pemetrexed, erlotinib, dasatanib,nilotinib, decatanib, panitumumab, amrubicin, oregovomab, Lep-etu,nolatrexed, azd2171, batabulin, ofatumumab, zanolimumab, edotecarin,tetrandrine, rubitecan, tesmilifene, oblimersen, ticilimumab,ipilimumab, gossypol, Bio 111, 131-I-TM-601, ALT-110, BIO 140, CC 8490,cilengitide, gimatecan, IL13-PE38QQR, INO 1001, IPdR₁ KRX-0402,lucanthone, LY 317615, neuradiab, vitespan, Rta 744, Sdx 102,talampanel, atrasentan, Xr 311, romidepsin, ADS-100380, sunitinib,5-fluorouracil, vorinostat, etoposide, gemcitabine, doxorubicin,liposomal doxorubicin, 5′-deoxy-5-fluorouridine, vincristine,temozolomide, ZK-304709, seliciclib; PD0325901, AZD-6244, capecitabine,L-Glutamic acid,N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-,disodium salt, heptahydrate, camptothecin, PEG-labeled irinotecan,tamoxifen, toremifene citrate, anastrazole, exemestane, letrozole,DES(diethylstilbestrol), estradiol, estrogen, conjugated estrogen,bevacizumab, IMC-1C11, CHIR-258);3-[5-(methylsulfonylpiperadinemethyl)-indolylj-quinolone, vatalanib,AG-013736, AVE-0005, the acetate salt of [D-Ser(But) 6, Azgly10](pyro-Glu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-Azgly-NH₂ acetate[C₅₉H₈₄N₁₈Oi₄-(C₂H₄O₂)_(x) where x=1 to 2.4], goserelin acetate,leuprolide acetate, triptorelin pamoate, medroxyprogesterone acetate,hydroxyprogesterone caproate, megestrol acetate, raloxifene,bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714;TAK-165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF antibody,erbitux, EKB-569, PKI-166, GW-572016, Ionafarnib, BMS-214662,tipifarnib; amifostine, NVP-LAQ824, suberoyl analide hydroxamic acid,valproic acid, trichostatin A, FK-228, SU11248, sorafenib, KRN951,aminoglutethimide, arnsacrine, anagrelide, L-asparaginase, BacillusCalmette-Guerin (BCG) vaccine, bleomycin, buserelin, busulfan,carboplatin, carmustine, chlorambucil, cisplatin, cladribine,clodronate, cyproterone, cytarabine, dacarbazine, dactinomycin,daunorubicin, diethylstilbestrol, epirubicin, fludarabine,fludrocortisone, fluoxymesterone, flutamide, gemcitabine, hydroxyurea,idarubicin, ifosfamide, imatinib, leuprolide, levamisole, lomustine,mechlorethamine, melphalan, 6-mercaptopurine, mesna, methotrexate,mitomycin, mitotane, mitoxantrone, nilutamide, octreotide, oxaliplatin,pamidronate, pentostatin, plicamycin, porfimer, procarbazine,raltitrexed, rituximab, streptozocin, teniposide, testosterone,thalidomide, thioguanine, thiotepa, tretinoin, vindesine,13-cis-retinoic acid, phenylalanine mustard, uracil mustard,estramustine, altretamine, floxuridine, 5-deooxyuridine, cytosinearabinoside, 6-mecaptopurine, deoxycoformycin, calcitriol, valrubicin,mithramycin, vinblastine, vinorelbine, topotecan, razoxin, marimastat,COL-3, neovastat, BMS-275291, squalamine, endostatin, SU5416, SU6668,EMD121974, interleukin-12, IM862, angiostatin, vitaxin, droloxifene,idoxyfene, spironolactone, finasteride, cimitidine, trastuzumab,denileukin diftitox, gefitinib, bortezimib, paclitaxel, cremophor-freepaclitaxel, docetaxel, epithilone B, BMS-247550, BMS-310705,droloxifene, 4-hydroxytamoxifen, pipendoxifene, ERA-923, arzoxifene,fulvestrant, acolbifene, lasofoxifene, idoxifene, TSE-424, HMR-3339,ZK186619, topotecan, PTK787/ZK 222584, VX-745, PD 184352, rapamycin,40-O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP-23573, RAD001,ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646,wortmannin, ZM336372, L-779,450, PEG-filgrastim, darbepoetin,erythropoietin, granulocyte colony-stimulating factor, zolendronate,prednisone, cetuximab, granulocyte macrophage colony-stimulating factor,histrelin, pegylated interferon alfa-2a, interferon alfa-2a, pegylatedinterferon alfa-2b, interferon alfa-2b, azacitidine, PEG-L-asparaginase,lenalidomide, gemtuzumab, hydrocortisone, interleukin-11, dexrazoxane,alemtuzumab, all-transretinoic acid, ketoconazole, interleukin-2,megestrol, immune globulin, nitrogen mustard, methylprednisolone,ibritgumomab tiuxetan, androgens, decitabine, hexamethylmelamine,bexarotene, tositumomab, arsenic trioxide, cortisone, editronate,mitotane, cyclosporine, liposomal daunorubicin, Edwina-asparaginase,strontium 89, casopitant, netupitant, an NK-1 receptor antagonists,palonosetron, aprepitant, diphenhydramine, hydroxyzine, metoclopramide,lorazepam, alprazolam, haloperidol, droperidol, dronabinol,dexamethasone, methylprednisolone, prochlorperazine, granisetron,ondansetron, dolasetron, tropisetron, pegfilgrastim, erythropoietin,epoetin alfa, darbepoetin alfa, ipilimumab, nivolomuab, pembrolizumab,dabrafenib, trametinib vemurafenib among others.

Pharmaceutical compositions according to the present invention comprisean effective amount of at least one ARM-U2 compound as otherwisedescribed herein, in combination with a pharmaceutically effectiveamount of a carrier, additive or excipient, optionally, in combinationwith one or more of the additional agents, especially anti-canceragents, otherwise described herein, all in effective amounts.

The ARM-U2 containing pharmaceutical compositions of the presentinvention may be formulated in a conventional manner using one or morepharmaceutically acceptable carriers and may also be administered inimmediate, early release or controlled-release formulations.Pharmaceutically acceptable carriers that may be used in thesepharmaceutical compositions include, but are not limited to, ionexchangers, alumina, aluminum stearate, lecithin, serum proteins, suchas human serum albumin, buffer substances such as phosphates, glycine,sorbic acid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as prolaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

The compositions of the present invention may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional and intracranial injection or infusiontechniques. Preferably, the compositions are administered orally,intraperitoneally or intravenously.

Sterile injectable forms of the compositions of this invention may beaqueous or oleaginous suspension. These suspensions may be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationmay also be a sterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilmay be employed including synthetic mono- or di-glycerides. Fatty acids,such as oleic acid and its glyceride derivatives are useful in thepreparation of injectables, as are natural pharmaceutically-acceptableoils, such as olive oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions may alsocontain a long-chain alcohol diluent or dispersant, such as Ph. Helv orsimilar alcohol.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, aqueous suspensions or solutions. In thecase of tablets for oral use, carriers which are commonly used includelactose and corn starch. Lubricating agents, such as magnesium stearate,are also typically added. For oral administration in a capsule form,useful diluents include lactose and dried corn starch. When aqueoussuspensions are required for oral use, the active ingredient is combinedwith emulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Alternatively, the pharmaceutical compositions of this invention may beadministered in the form of suppositories for rectal administration.These can be prepared by mixing the agent with a suitable non-irritatingexcipient which is solid at room temperature but liquid at rectaltemperature and therefore will melt in the rectum to release the drug.Such materials include cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions of this invention may also beadministered topically. Suitable topical formulations are readilyprepared for each of these areas or organs. Topical application for thelower intestinal tract can be effected in a rectal suppositoryformulation (see above) or in a suitable enema formulation.Topically-acceptable transdermal patches may also be used.

For topical applications, the pharmaceutical compositions may beformulated in a suitable ointment containing the active componentsuspended or dissolved in one or more carriers. Carriers for topicaladministration of the compounds of this invention include, but are notlimited to, mineral oil, liquid petrolatum, white petrolatum, propyleneglycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax andwater. In certain preferred aspects of the invention, the topical creamor lotion may be used prophylatically to prevent infection when appliedtopically in areas prone toward virus infection. In additional aspects,the compounds according to the present invention may be coated onto theinner surface of a condom and utilized to reduce the likelihood ofinfection during sexual activity.

Alternatively, the pharmaceutical compositions can be formulated in asuitable lotion or cream containing the active components suspended ordissolved in one or more pharmaceutically acceptable carriers. Suitablecarriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutical compositions may be formulated asmicronized suspensions in isotonic, pH adjusted sterile saline, or,preferably, as solutions in isotonic, pH adjusted sterile saline, eitherwith our without a preservative such as benzylalkonium chloride.Alternatively, for ophthalmic uses, the pharmaceutical compositions maybe formulated in an ointment such as petrolatum.

The pharmaceutical compositions of this invention may also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

The amount of compound in a pharmaceutical composition of the instantinvention that may be combined with the carrier materials to produce asingle dosage form will vary depending upon the host and diseasetreated, the particular mode of administration. Preferably, thecompositions should be formulated to contain between about 0.05milligram to about 1 to several grams, more preferably about 1 milligramto about 750 milligrams, and even more preferably about 10 milligrams toabout 500-600 milligrams of active ingredient, alone or in combinationwith at least one other ARM-U2 compound according to the presentinvention or other anti-cancer agent which may be used to treat canceror a secondary effect or condition thereof.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease or condition beingtreated.

A patient or subject (e.g. a male or female human) suffering from cancercan be treated by administering to the patient (subject) an effectiveamount of the ARM-U2 compound according to the present inventionincluding pharmaceutically acceptable salts, solvates or polymorphs,thereof optionally in a pharmaceutically acceptable carrier or diluent,either alone, or in combination with other known pharmaceutical agents,preferably agents which can assist in treating cancer and/or secondaryeffects of cancer or ameliorate the secondary effects and conditionsassociated with cancer, including metastasis of cancer. This treatmentcan also be administered in conjunction with other conventional cancertherapies, including radiation therapy.

These compounds can be administered by any appropriate route, forexample, orally, parenterally, intravenously, intradermally,subcutaneously, or topically, in liquid, cream, gel, or solid form, orby aerosol form.

The active compound is included in the pharmaceutically acceptablecarrier or diluent in an amount sufficient to deliver to a patient atherapeutically effective amount for the desired indication, withoutcausing serious toxic effects in the patient treated. A preferred doseof the active compound for all of the herein-mentioned conditions is inthe range from about 10 ng/kg to 300 mg/kg, preferably about 0.1 to 100mg/kg per day, more generally 0.5 to about 25 mg per kilogram bodyweight of the recipient/patient per day. A typical topical dosage willrange from 0.01-5% wt/wt in a suitable carrier.

The compound is conveniently administered in any suitable unit dosageform, including but not limited to one containing less than 1 mg, 1 mgto 3000 mg, preferably about 5 to 500-600 mg or more of activeingredient per unit dosage form. An oral dosage of about 25-250 mg isoften convenient.

The active ingredient is preferably administered to achieve peak plasmaconcentrations of the active compound of about 0.00001-30 mM, preferablyabout 0.1-30 μM. This may be achieved, for example, by the intravenousinjection of a solution or formulation of the active ingredient,optionally in saline, or an aqueous medium or administered as a bolus ofthe active ingredient. Oral administration is also appropriate togenerate effective plasma concentrations of active agent.

The concentration of active compound in the drug composition will dependon absorption, distribution, inactivation, and excretion rates of thedrug as well as other factors known to those of skill in the art. It isto be noted that dosage values will also vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that the concentration ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed composition. The active ingredient may be administered atonce, or may be divided into a number of smaller doses to beadministered at varying intervals of time.

Oral compositions will generally include an inert diluent or an ediblecarrier. They may be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound or its prodrug derivative can be incorporated with excipientsand used in the form of tablets, troches, or capsules. Pharmaceuticallycompatible binding agents, and/or adjuvant materials can be included aspart of the composition.

The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a bindersuch as microcrystalline cellulose, gum tragacanth or gelatin; anexcipient such as starch or lactose, a dispersing agent such as alginicacid, Primogel, or corn starch; a lubricant such as magnesium stearateor Sterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring. When the dosage unitform is a capsule, it can contain, in addition to material of the abovetype, a liquid carrier such as a fatty oil. In addition, dosage unitforms can contain various other materials which modify the physical formof the dosage unit, for example, coatings of sugar, shellac, or entericagents.

The active compound or pharmaceutically acceptable salt thereof can beadministered as a component of an elixir, suspension, syrup, wafer,chewing gum or the like. A syrup may contain, in addition to the activecompounds, sucrose as a sweetening agent and certain preservatives, dyesand colorings and flavors.

The active compound or pharmaceutically acceptable salts thereof canalso be mixed with other active materials that do not impair the desiredaction, or with materials that supplement the desired action, such asother anticancer agent, anti-HIV agents, antibiotics, antifungals,anti-inflammatories, or antiviral compounds. In certain preferredaspects of the invention, one or more ARM-U2 compounds according to thepresent invention are coadministered with another anticancer agentand/or another bioactive agent, as otherwise described herein.

Solutions or suspensions used for parenteral, intradermal, subcutaneous,or topical application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. The parental preparationcan be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic.

If administered intravenously, preferred carriers are physiologicalsaline or phosphate buffered saline (PBS).

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art.

Liposomal suspensions may also be pharmaceutically acceptable carriers.These may be prepared according to methods known to those skilled in theart, for example, as described in U.S. Pat. No. 4,522,811 (which isincorporated herein by reference in its entirety). For example, liposomeformulations may be prepared by dissolving appropriate lipid(s) (such asstearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline,arachadoyl phosphatidyl choline, and cholesterol) in an inorganicsolvent that is then evaporated, leaving behind a thin film of driedlipid on the surface of the container. An aqueous solution of the activecompound are then introduced into the container. The container is thenswirled by hand to free lipid material from the sides of the containerand to disperse lipid aggregates, thereby forming the liposomalsuspension.

General Chemical Synthesis

All ARM U2 derivatives may be synthesized by performing a highlygeneral, optimized nucleophillic substitution/deprotection reactionsequence carried out between the ABT variant of choice and amono-bromoisoxazole-anthroquinone core intermediate consisting ofvarious functionalized aniline derivatives all substituted at position 4of ring C (See FIG. 4, Scheme 1, FIG. 5, FIG. 6, FIG. 8, Table 1 and theexamples section of the present application). Isoxazole formation iscritical for the substitution of nucleophillic substituents at positions2 and 4 of ring C by disrupting ring C aromaticity affording a higherdegree of 1.4 michael acceptor character in ring C. Other compoundswhich are disclosed are synthesized by analogy using standard methodswhich are readily available in the art. It is noted that the isoxazolegroup may be readily cleaved to the corresponding keto and free aminefunctionality in certain compounds according to the present invention byexposing the compound to light or aqueous conditions (describedhereinbelow). These compounds, readily prepared, are also biologicallyactive.

The A_(B)Ms (see FIG. 4, scheme 1 and the examples section) employed inthis study were synthesized from commercially available polyethyleneglycol (PEG) derivatives of varying lengths, tethered to DNP on oneterminus and terminating in an L- or D-nipecotic amide residue servingas the nucleophile in the substitution reaction. Other linkers may bereadily substituted for the PEG linkers as otherwise described herein.Preceding the mono-bromoisoxazole core intermediate III (SI) in thesynthetic pathway (FIG. 4, Scheme 1) is a di-brominatedisoxazole-anthroquinone intermediate II (FIG. 4, Scheme 1) central tothe synthesis of all ARM-U2 derivatives synthesized which wassubsequently functionalized with various aniline derivatives throughselective lewis acid or metal mediated couplings to yield the respectiveARM-U2 derivative prior to the ABT substitution step.

Coupling the dibrominated isoxazole core with 3-aminobenzoate usingaluminum trichloride facilitates selective coupling of the anilinederivative at the 4-position over the 2-position of ring C and affordedthe key intermediate to access derivatives 1-ABT-1 and 1-D-ABT-1differing in the stereochemistry of the appended ABT. The di-acidderivative 2-ABT-1 could be efficiently accessed via aluminum chloridemediated coupling of aniline dibenzoate to intermediate II. ARM U2derivative 3-ABT-1 was efficiently accessed through facile coupling ofester-protected glycine to derivative 1-ABT-1. Derivative 4-ABT-1 withthe carboxyl extended farther from the aniline moiety was successfullysynthesized via a novel application of a modified Ullman-type coppercoupling reaction between methylester protected glycine and3-bromoaniline. Other amino acids may be used in place of the glycinewith appropriate protection groups being used.

The resulting glycine functionalized aniline could be coupledselectively to position 4 of intermediate II again by employing Lewisacid catalysis with aluminum chloride The mono-meta sulfonate derivative5-ABT-1 was afforded via an adapted aluminum chloride mediate couplingprocedure linking metanilic acid to the 4-position of ring C ofintermediate II. Solubilization of metanilic acid with careful controlof DIPEA equivalents proved critical for a high yielding reaction. Thedi-sulfonate derivative 6-ABT-1 was synthesized by a highly optimizedLi₂CO₃ mediated, Cu(II)OAc catalyzed coupling reaction betweenaniline-2,4-disulfonic acid and intermediate II. Aluminum trichloridemediates coupling of the sulfonated aniline was unsuccessful likely dueto deactivation of the aniline by ortho and para sulfonates as well aspotential sulfaonte chelation to the catalyst. The reaction provedhighly sensitive to temperature, organic bases, copper II equivalentsand requires a large excess of the aniline for conversion to producthowever achieves selective coupling for the 4-position over the2-position of ring C.

Longer ABT containing ARM-U2 derivatives 1-ABT-2, 1-ABT-3, 2-ABT-26-ABT-2 and 6-ABT-3 were also synthesized using identical chemistry astheir shorter linker ABT-1 counterparts only differing in theincorporation of longer PEG-derived spacer units Control ARM-U2derivative 6-ABT-4 lacking antibody recruiting capability due to a DNPto pyrimidine substitution was synthesized as described above for6-ABT-1 only incorporating an additional copper catalyzed coupling stepto install the pyrimidine unit onto the linker.

Each of the ARM-U2 derivatives proposed by docking studies weresynthesized initially on a ten milligram scale and can be successfullyscaled up to 0.5 g. Each derivative was purified by HPLC as the finalpurification step using formic acid buffers or in the special case of6-ABT-1/2/3 using ammonium formate buffers and were successfullycharacterized by proton and carbon NMR and high resolution ESI/MS.

The scheme set forth in FIG. 5 hereof sets forth the synthesis ofcompound ARM-US 0040, a preferred compound. Formation of tricycliccompound 1 proceeds by reacting the commercially available diene withthe bicyclic dione compound. The hydroxyl group of compound I isconverted to an amine group to form compound II, which is subsequentlyreacted to form the dibromo derivative III. The amine and keto group iscyclized to form the dibromo isoxazole compound IV which is then reactedwith the di-sulfonic acid aniline intermediate to form compound V.Compound V is then condensed with the piperidine linked dinitrophenolfunctional linking group, displacing the bromo substituent to form thefinal ARM-U2 0040 compound.

Other compounds which are described in the present application arereadily synthesized by modifying the synthetic steps which are describedabove or made by analogy following routing synthetic chemical syntheticsteps well known in the arts.

In the case of ring opened compounds (where the isoxazole moiety isring-opened and replaced by a keto group and amine group as set forthherein) as described in the present invention, the preferred approachfor opening the isoxazole ring is to expose the isoxazole compound insolvent to light at room temperature or in certain instances, elevatedtemperature, or alternatively, the compound may be dissolved in anaqueous solvent to provide an aqueous solution which can be stirred atroom temperature or reduced or elevated temperature in order to reduceto increase the formation of the ring-opened compound. The ring-openedcompounds exhibit biological activity similar to the isoxazole compoundsin the nanomolar to micromolar range. The following examples aredescribed in order to provide specific chemical synthetic proceduresand/or biological activities.

Examples

The following chemical examples are provided. Methodology and datarelated to chemistry and biology is included here and/or in the attachedAppendices A and/or B.

General Information

Synthesis: All reagents were purchased from commercial suppliers andused without further purification except the following: triethylaminewas distilled over calcium hydride; CH₂Cl₂, PhMe, DMF, and THF werepurified using a solvent dispensing system;¹ Water was purified using aMilli-Q purification system. Infrared (IR) spectra bands arecharacterized as broad (br), strong (s), medium (m) and weak (w). ¹H NMRchemical shifts are reported with the solvent residual peak as theinternal standard (CDCl₃ 7.26 ppm or DMSO 3.31 ppm). Data are reportedas follows: chemical shift, integration, multiplicity (s=singlet,d=doublet, t=triplet, q=quartet, br=broad, m=multiplet), and couplingconstants (Hz). ¹³C NMR chemical shifts are reported in ppm with thesolvent as an internal reference (CDCl₃ 77.2 ppm or CD₃OD 49.00 ppm ord-DMSO 39.5 ppm). Polyethylene glycol linker-based derivatives(C₂H₂O)_(n)H were synthesized from the corresponding polyethylene glycolof the highest oligomer purity commercially available through Aldrich(all cases >90% major oligomer).

Biology: A172 human glioblastoma cells were purchased from ATCC(#CRL-1620), grown in T-flasks with Dulbecco's modified Eagle's mediumsupplemented with 10% HI-FBS, and detached by the EDTA detachmentprocedure. U937 Cells were purchased from ATCC (#CRL-1593.2), grown inPetri dishes as a suspension with RPMI-1640 medium supplemented with 10%HI-FBS and 1% penicillin-streptomycin. All cell culturing was done usingcolored ADCP Media RPMI Medium 1640, liquid Invitrogen #11875-093supplemented with 10% HI-FBS and 1% penicillin-streptomycin.Anti-Dinitrophenyl-KLH Rabbit IgG Fraction with and without biotin werepurchased from Invitrogen #A6430 (Lot 807872) as a solution and storedat 4° C. Human uPAR Antibody: Polyclonal goat IgG R&D Systems #AF807 waspurchased as a lyophilized solid and was stored at −20° C., as asolution in 250 μL of sterile DPBS. Human urokinase, isolated from humanurine was purchased from PROSPEC as a lyophilized white solid andreconstituted at a concentration of 1 mg/ml (approx. 18.5 uM) in milliQwater and stored at −20° C. Recombinant human uPAR with carrier proteinwas obtained as a white lyophilized solid from R&D systems and dissolvedin DPBS at a concentration of 100 ug/ml (approx. 2 uM) and stored at−20° C.

Synthesis

Synthesis of Intermediate I

To a flame dried flask under argon was added5-hydroxy-1,4-naphthoquinone (300 mg, 1.7 mmol) followed by 30 ml ofanhydrous DCM. The flask was flushed with argon followed by the additionof 1-(trimethylsiloxy)-1,3-butadiene (0.447 ml, 2.55 mmol) by syringeand left to stir at RT for 24 hr under argon. Upon complete consumptionof starting material, 1.2 ml of anhydrous triethylamine was added andleft to stir at RT for 12 hr. The solution was extracted against DCM andbrine/1N HCL, dried using sodium sulfate, and purified by columnchromatography (1:1 Hex/DCM to 100% DCM resulting in the isolation ofpure hydroxyanthroquinone (intermediate i) in 47% yield (180 mg, 0.8mmol) as a yellow solid. ¹H NMR (600 MHz, Chloroform-d) 12.60 (s, 1H),8.30 (m, 2H), 7.82 (m, 3H), 7.67 (t, J=7.9 Hz, 1H), 7.31 (d, J=8.3 Hz,1H). ¹³C NMR (151 MHz, CDCl₃) 188.65, 182.41, 162.54, 136.74, 134.66,134.17, 133.59, 133.43, 133.17, 127.41, 126.90, 124.35, 119.55, 116.13.HRMS (ES+) calc'd for C₁₄H₉O₃ (M+H) m/z 225.0473, Found. 225.0461 Theanthroquinone (0.180 g, 0.8 mmol) was dissolved in 10 ml anhydrous DMFto which KI (13 mg, 0.08 mmol), potassium carbonate (221 mg, 1.6 mmol),and chloroacetamide (93 mg, 1.0 mmol) were added. The solution washeated to 90° C. for 1 hr followed by 150° C. for 24 hours followed byextraction with 1N HCL and EtOAc. The organic fractions were dried usingsodium sulfate, concentrated in vacuo, and the remaining red residuepurified by ISCO silica chromatography using a Hex/EtOAc gradient (10%EtOAc to 50% over 30 min) resulting in the isolation ofanilinoanthroquinone intermediate ii (156 mg, 0.7 mmol) in 87% yield asa red solid ¹H NMR (600 MHz, Chloroform-d) 8.29 (d, J=7.7 Hz, 1H), 8.25(d, J=7.6 Hz, 1H), 7.77 (t, J=7.5 Hz, 1H), 7.72 (t, J=7.5 Hz, 1H), 7.65(d, J=7.3 Hz, 1H), 7.46 (t, J=7.8 Hz, 1H), 6.97 (d, J=8.3 Hz, 1H), 6.84(brm, 2H). ¹³C NMR (151 MHz, CDCl₃) 185.24, 183.60, 150.99, 134.73,134.40, 133.94, 133.17, 129.64, 128.31, 126.79, 123.08, 117.29, 113.65.HRMS (ES+) calc'd for C₁₄H₉NO₂ (M+H) m/z 224.0633, Found. 224.0623.

The anilinoanthroquinone (78 mg, 0.35 mmol) was dissolved in 5 ml ofglacial acetic acid predried using 3 A molecular sieves to which brominewas added (72 ul, 1.4 mmol) and the reaction stirred at room temperaturefor 12 hr. The solution was extracted against DCM and brine, the organiclayers combined, dried, and concentrated as described above with theisolated red solid purified by ISCO silica chromatography using ahexane/DCM gradient (20% DCM-100% DCM). The isolated fractions wereconcentrated with the crude brominated anthroquinone productcrystallized out of DCM/MeOH resulting in the isolation of puredibromo-anilinoanthroquinone I in 65% yield ¹H NMR (600 MHz,Chloroform-d) 8.24 (td, J=7.5, 2.2 Hz, 2H), 8.08 (s, 1H), 7.76 (ddd,J=6.4, 3.8, 1.8 Hz, 2H). ¹³C NMR (151 MHz, CDCl₃) 184.37, 182.27,147.97, 143.97, 133.94, 133.82, 133.45, 133.26, 130.67, 127.08, 126.60,117.50, 115.33, 108.71. HRMS (ES+) calc'd for C₁₄H₈Br₂NO₂ (M+H) m/z379.8844 Found. 379.8841.

Synthesis of ARM-U2 Derivative 3-(R)-ABT-1

The synthesis of intermediate III was carried out as describedpreviously (REF). Briefly, 1-amino-2,4-dibromoanthraquinone (I) (5 g,13.1 mmol), was subjected to diazotization in the presence of sodiumnitrate and sulfuric acid. Following displacement by aqueous sodiumazide, cyclization to furnish the isoxazole was accomplished byrefluxing in toluene to yield intermediate II in 66% yield as reportedpreviously (REF). The di-bromo-isoxazole Intermediate II (200 mg, 0.52mmol) was coupled to methyl-3-aminobenzoate in nitrobenzene using AlCl₃to furnish the aniline substituted derivative III in 60% yield asreported previously (REF). The first and shortest length PEG derivedantibody binding terminus 1 (S/R)-ABT-1 synthesized in this report, wasprepared by first coupling 1-chloro-2,4-dinitrobenzene to4,7,10-trioxa-1,13-tridecanediamine as described previously (REF). Theresulting DNP appended PEG-linker (1.16 g, 3 mmol) was subsequentlydissolved in 20 ml DCM to which BOC-D-nipecotic acid or BOC-L-nipecoticacid (0.5 g, 2.2 mmol), EDC (0.506 g, 2.64 mmol), and HOBt were added.The solution was left stirring at room temperature overnight. Thesolution was then diluted with 50 ml DCM and washed with a 1:1:1 mixture(60 ml) of water, brine, and saturated sodium bicarbonate (3×) followedby a 1:1:1 mixture (60 ml) of 10% citric acid, water, and brine. Theorganic layer was dried using sodium sulfate, filtered, and concentratedresulting in a dark-reddish oil. The crude BOC-protected (R/S)-ABT-1,was dissolved in a 1:1 mixture of TFA/DCM and stirred at roomtemperature for 5 hr. TFA was removed under a stream of nitrogen and theDCM was removed in vacuo. The dark oil was dissolved in EtOAc, washedwith a 1:1:1 solution of 10% NaOH/water/brine, dried with sodium sulfateand concentrated to yield crude R/S-ABT-1 (1.3 g, quantitative) useddirectly in the next step. R-ABT-1 (31 mg, 0.0625 mmol) was dissolved in1 ml of anhydrous ACN to which dry DIPEA (0.31 mmol) and intermediateIII were added (11 mg, 0.025 mmol). The reaction was allowed to proceedfor 5 hr at 80° C. in the dark with quantitative conversion to productconfirmed by LC-MS. To the resulting orange-reddish solution was added 2ml of 10% aqueous NaOH and 1 ml methanol and the solution was leftstirring for 5 hr at room temperature in the dark. Followingconfirmation of quantitative ester deprotection by LC-MS, a stream ofnitrogen was passed over the solution to remove the majority of organicsolvent, and the resulting slurry dissolved in DMSO. The product waspurified by ISCO C-18 reverse-phase chromatography (shielded from directlight) with a gradient of 30% ACN-100% ACN over 50 min (Both water andACN contain 10 mM pH 4 ammonium formate buffer) and followinglyophillzation yielded 1-(R)-ABT-1 in 44% yield (9.5 mg, 0.011 mmol) asa lyophilized reddish-orange solid. ¹H NMR (500 MHz, DMSO-d₆) 11.76 (s,1H), 8.88 (t, J=5.5 Hz, 1H), 8.73 (d, J=2.7 Hz, 1H), 8.43 (d, J=7.9 Hz,1H), 8.17-8.1 (m, 2H), 7.99 (s, 1H), 7.91 (br s, 1H) 7.82 (m, 2H), 7.70(m, 2H), 7.58 (t, J=7.8 Hz, 1H), 7.09 (d, J=9.7 Hz, 1H), 6.38 (s, 1H),4.55 (br s, 2H), 4.37 (br s, 2H) 3.55-3.3 (m, 14H), 3.19-2.98 (m, 2H),2.80 (m, 1H), 1.84 (m, 4H), 1.58 (m, 4H). ¹³C NMR (151 MHz, DMSO)175.50, 172.43, 167.28, 154.29, 153.21, 148.49, 146.19, 138.59, 134.92,133.25, 131.86, 130.41, 130.24, 129.81, 128.98, 127.95, 126.53, 123.98,123.95, 123.91, 122.32, 118.98, 115.36, 95.83, 94.71, 70.15, 69.93,68.72, 68.44, 55.36, 41.43, 40.86, 36.16, 29.61, 28.65, 27.86. HRMS(ES+) calc'd for C₄₃H₄₆N₇O₁₂ (M+H) m/z 852.3204, Found. 852.3296.

Synthesis of ARM-U2 Derivative 1-ABT-1

ARM-U2 derivative 1-ABT-1 was prepared and purified identically asdescribed for 1-(R)-ABT-1 only ABT-1 was synthesized usingBoc-L-nipecotic acid. 1-ABT-1 was isolated in 52% yield (11 mg, 0.013mmol). ¹H NMR (500 MHz, DMSO-d₆) 11.78 (s, 1H), 8.89 (t, J=5.6 Hz, 1H),8.74 (d, J=2.7 Hz, 1H), 8.44 (d, J=8.0 Hz, 1H), 8.34 (s, 1H), 8.13 (m,2H), 8.05 (s, 1H), 7.82 (t, J=8.1 Hz, 1H), 7.77 (d, J=6.9 Hz, 1H), 7.69(t, J=8.1 Hz, 1H), 7.49 (m, 2H), 7.10 (d, J=9.7 Hz, 1H), 6.43 (s, 1H),4.55 (br s, 2H), 4.35 (br s, 2H) 3.57-3.29 (m, 14H), 3.08 (m, 2H), 2.60(t, J=10.7 Hz, 1H), 1.84 (m, 4H) 1.54 (m, 4H). ¹³C NMR (151 MHz, DMSO)174.92, 171.96, 167.95, 164.59, 153.70, 152.84, 148.08, 145.53, 137.49,134.47, 132.84, 131.34, 129.83, 129.36, 128.46, 127.51, 126.06, 123.50,123.28, 121.87, 118.56, 114.97, 95.37, 94.51, 69.74, 69.50, 68.31,68.11, 54.99, 41.02, 39.94, 35.82, 29.16, 28.25, 27.56. HRMS (ES+)calc'd for C₄₃H₄₆N₇O₁₂ (M+H) m/z 852.3204. Found. 852.3390.

Synthesis of PEG-8 Derived (R/S)-ABT-2

ABT-2 (Scheme S2) was synthesized from commercially availableoctaethylene glycol. The diol (5 g, 0.0135 moles) was dried for 24 hoursunder vacuum while stirring with molecular sieves prior to use followingwhich time, it was dissolved in anhydrous DCM (60 ml) under nitrogen. Tothis solution was added dry Et₃N (4.5 ml, 0.033 moles) followed by TsCl(7.0 g, 0.04 moles) and finally DMAP (0.1645 g, 0.00135 moles). Thereaction was allowed to proceed for 12 hours at room temperature atwhich time the solution was washed with 1N HCl followed by an aqueoussaturated sodium bicarbonate solution. The organic layers were combined,dried using sodium sulfate, concentrated in vacuo and the resulting oilpurified by ISCO silica chromatography with a DCM/MeOH gradient.Bis-tosyl PEG-8 was isolated as a pale yellow oil in 89% yield (8 g,0.012 moles). Bis-tosyl PEG-8 (2 g, 0.0029 moles) was dissolved in 24 mlof a 1:1 DMF/DMSO solution to which NaI (0.153 g, 0.001 moles) and NaN₃(1.34 g, 0.021 moles) were added. The solution was left stirring at 60°C. over night, diluted in diethylether and extracted against brine andsodium bicarbonate. The organic layers were combined, dried using sodiumsulfate and concentrated in vacuo to yield bis-azide PEG-8 in approx.80% crude yield (1.7 g, 0.004 moles). The bis-azide (1.2 g, 0.0028moles) was dissolved in 46 ml of methanol and reduced under anatmosphere of hydrogen in the presence of 10% Pd/C. The reaction mixturewas filtered through a celite plug, eluted with methanol, andconcentrated to yield PEG8-diamine containing a significant amount ofNaOMe but with sufficient purity to functionalize with DNP chloride.Peg8-diamine was acidified using 4M HCL in dioxane and dried in vacuo toyield PEG8-diamine in 95% yield as the HCL salt (1 g, 0.0027 moles). ThePEG8-diamine HCL salt (0.846 g, 0.0023 moles) was neutralized with 1 eqDIPEA and refluxed in 10 ml of EtOH to which small portions of DNP-Cl(0.465 g, 0.0023 moles total) were added over 5 hr, diluted in DCM andwashed with sodium bicarbonate and brine. Key intermediate iv wasisolated as reddish oil in 69.5% yield (0.860 g, 0.0016 moles) andcoupled directly to L or D-nipecotic acid (0.550 g, 0.0024 moles) in 50ml DMF in the presence of DIEPA (418 ul, 0.0024 moles) and HATU (0.912g, 0.0024 moles) for 5 hr at room temperature. The solution wasextracted against using brine/EtOAc and the organic layer concentratedin vacuo. The dark reddish oil was dissolved in 4M HCl/dioxane andstirred for 5 hr at room temperature following which time the solutionwas concentrated to dryness under a stream of nitrogen and leftovernight under vacuum. Crude (R or S) ABT-2 was purified using HPLC(water/ACN 0.1% formic acid) to yield pure R or S-ABT-2 as the formatesalt in 75% yield over 2 steps (0.64 g, 0.0012 moles). S-isomer: ¹H NMR(600 MHz, Chloroform-d) 9.13 (d, J=2.6 Hz, 1H), 8.81 (m, 1H), 8.27 (d,J=12.0 Hz, 1H), 7.55 (m, 1H), 6.97 (d, J=9.5 Hz, 1H), 3.83-3.53 (m,32H), 3.44 (m, 1H), 3.39 (m, 2H), 3.29 (m, 1H), 3.12 (m, 1H), 2.97 (s,1H), 2.90 (m, 1H), 1.95 (m, 1H), 1.90 (m, 2H), 1.81 (m, 1H). ¹³C NMR(151 MHz, CDCl₃) 172.38, 148.41, 136.03, 130.27, 124.28, 114.16, 70.28,69.48, 68.53, 50.66, 45.95, 43.90, 43.22, 39.18, 38.94, 25.96, 21.05.HRMS (ES+) calc'd for C₂₈H₄₇N₅O₁₂ (M+H) m/z 645.3221. Found. 645.3207.

Synthesis of ARM-U2 Derivative 1-(R)-ABT-2

The synthesis and purification of ARM-U2 derivative 1-(R)-ABT-2 wascarried out exactly as described above for derivative 1-(R)-ABT-1 exceptthat (R)-ABT-2 (48 mg, 0.075 mmol) was coupled to intermediate III asopposed to intermediate (R)-ABT-1 to yield 1-(R)-ABT-2 in 50% yield(12.5 mg, 0.0125 mmol). ¹H NMR (500 MHz, DMSO-d₆) 11.78 (s, 1H),8.83-8.75 (m, 2H), 8.45 (d, J=8.0 Hz, 1H), 8.17 (m, 2H), 8.08 (br s,1H), 8.00 (s, 1H) 7.82-7.79 (m, 2H), 7.75-7.66 (m, 2H), 7.58 (t, J=7.8Hz, 1H), 7.20 (d, J=9.6 Hz, 1H), 6.40 (s, 1H), 4.51 (br s, 4H),3.72-3.11 (m, 32H), 2.65-2.56 (m, 1H), 1.88 (m, 1H), 1.76 (m, 1H),1.67-1.57 (m, 1H), 1.24 (m, 1H). ¹³C NMR (126 MHz, DMSO) 175.50, 172.72,167.45, 154.28, 153.24, 148.71, 148.52, 146.19, 138.50, 135.24, 133.27,131.85, 130.31, 130.19, 129.97, 129.54, 129.14, 128.96, 127.97, 126.52,123.97, 123.94, 123.86, 122.31, 119.00, 115.97, 95.85, 94.82, 70.23,70.04, 69.38, 68.65, 55.36, 43.08, 42.34, 38.91, 27.98, 24.41. HRMS(ES+) calc'd for C₄₃H₄₅N₇O₁₂ (M+H) m/z 852.3126. Found. 852.3156.

Synthesis of ARM-U2 Derivative 1-ABT-2

ARM-U2 derivative 1-ABT-2 was synthesized and purified as describedabove for 1-ABT-1 only ABT-2 was coupled to intermediate III as opposedto ABT-1 resulting in the isolation of 1-ABT-2 in % 45 yield (11.25 mg,0.011 mmol) ¹H NMR (500 MHz, DMSO-d₆) 11.81 (s, 1H), 8.80 (m, 1H), 8.47(d, J=8.0 Hz, 1H), 8.25 (s, 1H), 8.22-8.14 (m, 2H), 8.08 (s, 1H), 7.84(t, J=7.3 Hz, 1H), 7.77 (d, J=6.9 Hz, 1H), 7.72 (t, J=7.5 Hz, 1H), 7.50(br m, 2H), 7.21 (d, 1H), 6.49 (s, 1H), 4.72 (br s, 2H) 4.36 (brs, 2H)3.70-3.13 (m, 32H), 2.66 (m, 1H), 1.93-1.77 (m, 3H), 1.66-1.56 (m, 1H).¹³C NMR (151 MHz, DMSO) 175.34, 172.63, 168.64, 165.35, 154.11, 153.39,148.71, 148.58, 145.97, 137.70, 135.22, 133.30, 131.78, 130.20, 129.96,129.57, 128.89, 127.96, 126.51, 123.95, 123.88, 122.30, 119.02, 115.99,95.78, 95.10, 70.18, 69.98, 69.25, 68.63, 43.06, 38.88, 28.06. HRMS(ES+) calc'd for C₄₉H₅₈N₇O₁₆ (M+H) m/z 1000.3940. Found. 1000.3959.

Synthesis of PEG-8 Derived ABT-3

Key intermediate iv (0.485 g, 0.0009 moles) described above was coupledto glutaric anhydride (0.155 g, 0.00136 moles) in 10 ml of anhydrous THFin the presence of DIPEA (236 ul, 0.0013 moles) for 8 hr at roomtemperature (Scheme S2). The solution was concentrated under reducedpressure and purified by ISCO-C18 chromatography (water/ACN 0.1%triethylamine) to obtain the crude DNP-PEG8-acid (0.57 g, 0.0008 moles)in approx. 88% yield. The DNP-PEG8-acid (0.57 g, 0.0008 moles) wasdissolved in 5 ml of anhydrous DMF and added portion wise to a solutionof PEG8diamine HCl (0.59 g, 0.0016 moles) HATU (0.304 g, 0.0008 moles),and anhydrous DIPEA (140 ul, 0.0008 moles) in 5 ml anhydrous DMF. Thesolution was purified by C18 ISCO using 0.1% formic acid/MeOH to yieldthe crude DNP-PEG16-amine in approx. 80% yield (0.639 g, 0.00064 moles).Boc-L-nipecotic acid coupling, deprotection and linker purification wasquantitative and carried out as described for the synthesis of ABT-2 toyield ABT-3 in 66% overall yield (0.658 g, 0.0006 moles). ¹H NMR (400MHz, DMSO-d₆) 8.87 (d, J=2.8 Hz, 1H), 8.49 (brs, 1H) 8.27 (dd, J=9.6,2.7 Hz, 1H), 7.93 (t, J=5.3 Hz, 1H), 7.85 (t, J=5.4 Hz, 2H), 7.29 (d,J=9.7 Hz, 1H), 3.69 (m, 6H), 3.57 (m, 2H), 3.50 (m, 48H), 3.38 (m, 4H),3.17 (m, 4H), 2.92-2.76 (m, 4H), 2.44 (m, 1H) 2.26-2.17 (m, 1H), 2.04(t, J=7.5 Hz, 4H), 1.75-1.64 (m, 2H), 1.57-1.42 (m, 2H), 1.37-1.28 (m,2H). ¹³C NMR (151 MHz, DMSO) 174.23, 172.22, 166.24, 148.82, 135.30,130.32, 124.00, 116.14, 70.20, 68.70, 48.93, 45.99, 43.09, 38.85, 38.73,35.11, 28.10, 25.21, 21.94. HRMS (ES+) calc'd for C₄₉H₈₈N₇O₂₁ (M+H)1109.5955 m/z. Found 1109.5932.

Synthesis of ARM-U2 Derivative 1-ABT-3

ARM-U2 derivative 1-ABT-3 was synthesized and purified as describedabove for 1-ABT-1 only ABT-3 (83 mg, 0.075 mmol) was coupled tointermediate III giving rise to 1-ABT-3 in 38% yield (13.9 mg, 0.0095mmol) ¹H NMR (500 MHz, DMSO-d₆) 11.82 (s, 1H), 8.82 (d, J=2.7 Hz, 1H),8.57 (brs, 1H), 8.47 (d, J=7.9 Hz, 1H), 8.44 (brs, 1H), 8.22 (dd, J=9.6,2.5 Hz, 1H), 8.18 (d, J=7.8 Hz, 1H), 8.10 (s, 1H), 7.86-7.82 (m, 3H),7.74 (d, J=7.4, 1H), 7.71 (m, 1H)), 7.41 (m, 1H), 7.25 (d, J=9.7 Hz,1H), 6.52 (s, 1H), 4.77 (brs, 2H), 4.19 (brs, 2H), 3.73-3.09 (m, 64H),2.67 (m, 1H), 2.03 (t, J=7.4 Hz, 4H), 1.91-1.78 (m, 3H), 1.67-1.58 (m,3H). ¹³C NMR (151 MHz, DMSO) 175.31, 172.56, 172.23, 168.67, 165.72,154.08, 153.44, 148.77, 148.63, 145.87, 137.43, 135.27, 133.33, 131.77,130.27, 130.03, 129.32, 128.88, 127.98, 126.45, 123.95, 123.78, 123.56,122.32, 119.05, 116.07, 95.80, 95.28, 70.12, 69.97, 69.55, 51.74, 50.85,43.08, 41.88, 38.85, 35.11, 28.11, 25.00, 21.94. HRMS (ES+) calc'd forC₇₀H₉₇N₉O₂₅ (M+H) 1464.6596 m/z. Found 1464.6433.

Synthesis of ARM-U2 Derivative 2-ABT-1

Derivative 2-ABT-1 (Scheme S3) was synthesized as described above for1-ABT-1 only differing in that dimethyl-5-aminoisophthalate was coupledto intermediate II instead of methyl-3-aminobenzoate to afford themono-brominated product. Briefly, intermediate II (200 mg, 0.526 mmol)was dissolved in dry nitrobenzene along withdimethyl-5-aminoisophthalate (665 mg, 3 mmol). To this stirred solutionwas added anhydrous AlCl₃ (0.353 g, 2.65 mmol) and the reaction left toproceed for 3 hr. The resulting red solid intermediate V, wasprecipitated from ice water, recrystallized out of toluene in 36% crudeyield (100 mg, 0.19 mmol) and carried directly over to the next step.The mono-brominated intermediate V (10 mg, 0.019 mmol) was coupled toABT-1, deprotected, and purified as described above for the synthesis ofderivative 1-ABT-1 resulting in the isolation of derivative 2-ABT-1 as ared solid in 40% yield (7 mg, 0.0076 mmol). ¹H NMR (500 MHz, DMSO-d₆)11.77 (s, 1H), 8.89 (t, J=5.3 Hz, 1H), 8.73 (d, J=2.6 Hz, 1H), 8.43 (d,J=8.0 Hz, 1H), 8.30 (s, 1H), 8.19-8.08 (m, 5H), 7.83 (t, J=7.5 Hz, 1H),7.70 (t, J=7.6 Hz, 1H), 7.09 (d, J=9.7 Hz, 1H), 6.43 (s, 1H), 4.49 (brs, 4H), 3.6-3.21 (m, 14H), 3.07 (m, 2H), 2.57 (m, 1H), 1.83 (m, 4H),1.59 (m, 4H). ¹³C NMR (126 MHz, DMSO) 175.62, 172.35, 166.96, 163.63,154.43, 152.92, 148.48, 146.02, 138.80, 134.93, 133.24, 131.88, 130.22,129.82, 129.00, 127.98, 126.82, 123.98, 123.88, 122.31, 118.99, 115.36,96.09, 94.87, 70.16, 69.90, 68.72, 68.50, 41.44, 36.24, 29.56, 28.66,27.93. HRMS (ES+) calc'd for C₄₄H₄₆N₇O₁₄ (M+H) 896.3103 m/z 896.3103.Found 896.3094.

Synthesis of ARM-U2 Derivative 2-ABT-2

The synthesis of ARM-U2 derivative 4-ABT-2 (Scheme S3) was carried outexactly as described above for derivative 2-ABT-1 except that linkerderivative ABT-2 was employed. Crude intermediate V (10 mg, 0.019 mmol)was coupled to derivative ABT-2, deprotected and purified as describedfor the synthesis of 2-ABT-1 giving rise to 2-ABT-2 in 43% yield (8.5mg, 0.0082 mmol). ¹H NMR (500 MHz, DMSO-d₆) 11.78 (s, 1H), 8.78 (m, 2H),8.44 (d, J=7.8 Hz, 1H), 8.31 (s, 1H), 8.16 (m, 5H), 7.85-7.80 (m, 1H),7.73-7.68 (m, 1H), 7.19 (d, J=9.6 Hz, 1H), 6.43 (s, 1H), 4.51 (br s,4H), 3.73-3.08 (m, 32H), 2.59 (m, 1H), 1.94-1.70 (m, 3H), 1.62 (m, 1H).¹³C NMR (126 MHz, DMSO) 175.64, 172.66, 166.90, 154.45, 152.92, 148.70,148.49, 146.08, 138.92, 135.24, 133.24, 131.91, 130.18, 129.96, 129.01,128.00, 126.96, 126.80, 124.00, 123.85, 122.32, 119.00, 115.96, 96.09,94.87, 70.02, 69.32, 68.64, 43.07, 42.9, 38.9, 28.00, 24.39. HRMS (ES+)calc'd for C₅₀H₅₇N₇O₁₈ (M+H) 1043.3760 m/z. Found. 1043.3748.

Synthesis of ARM-U2 Derivative 3-ABT-1

ARM-U2 derivative 3-ABT-1 was synthesized by coupling derivative 1-ABT-1(10 mg, 0.011 mmol) to GlyOMe (2.5 mg, 0.022 mmol) in the presence ofDIPEA (2.3 ul, 0.0132 mmol), EDC (2.5 mg, 0.0132 mmol) and HOBt (1.8 mg,0.0132 mmol) in DCM (2 ml) for 12 hr at room temperature (Scheme S4-A).The solution was diluted with 10 ml of DCM and washed 3× with 10 ml ofbrine. The organic layers were combined, dried with sodium sulfate andconcentrated. The reddish residue was dissolved in a 1:1:1 solution ofTHF/MeOH/10% NaOH and stirred at room temperature for 5 hr. Organicswere then removed under a stream of nitrogen and the remainingprecipitate dissolved in DMSO and purified as described for ARM-U21-ABT-1 yielding 3-ABT-1 in 83% yield (8.2 mg, 0.009 mmol). ¹H NMR (500MHz, DMSO-d₆) 11.81 (s, 1H), 8.90 (t, J=5.6 Hz, 1H), 8.78 (m, 1H), 8.75(d, J=2.7 Hz, 1H), 8.45 (d, J=8.0 Hz, 1H), 8.17-8.13 (m, 2H), 7.98 (s,1H), 7.91 (t, J=5.3 Hz, 1H), 7.83 (t, J=7.0 Hz, 1H), 7.77 (d, J=7.7 Hz,1H), 7.71 (t, J=7.6 Hz, 1H), 7.60. (t, J=7.8 Hz, 1H), 7.12 (d, J=9.7 Hz,1H), 6.37 (s, 1H), 4.53 (br s, 2H), 4.39 (br s, 2H), 3.89-3.85 (m, 2H),3.56-3.33 (m, 14H), 3.14 (m, 2H), 2.63 (m, 1H), 1.88 (m, 4H), 1.59 (m,4H). ¹³C NMR (126 MHz, DMSO) 175.48, 172.51, 171.46, 165.65, 154.27,153.33, 148.51, 146.28, 138.58, 136.15, 134.95, 133.27, 131.84, 130.35,130.27, 129.86, 129.53, 128.96, 127.96, 126.29, 124.68, 123.92, 122.27,118.97, 115.39, 95.78, 94.76, 70.15, 69.94, 68.73, 68.43, 42.74, 42.52,41.45, 36.14, 29.64, 28.66, 27.83. HRMS (ES+) calc'd for C₄₅H₄₉N₈O₁₃(M+H) m/z 909.3419.

Found. 909.3478.

Synthesis of ARM-U2 Derivative 4-ABT-1

To a flame dried flask was added CuI (10 mg, 0.05 mmol), K₃PO₄ (636 mg,3 mmol), N,N-diethylsalicylamide (39 mg, 0.2 mmol), and GlyOMe. HCl (188mg, 1.5 mmol). The flask was backflushed with argon followed by theaddition of anhydrous DMF (1 ml), and 3-bromoaniline (108 ul, 1 mmol)(Scheme S4-B). The mixture was stirred at 90° C. for 12 hr under argon,diluted with EtOAc and washed with brine 3× and saturated sodiumbicarbonate. The EtOAc layers were collected dried with sodium sulfateand concentrated. The remaining oil was purified by ISCO flashchromatography with a 12 g column using a 0-50% gradient of DCM/EtOAcover 35 min. Pure glycine ester coupled aryl product intermediate VI wasobtained in 10% yield (20 mg, 0.1 mmol). ¹H NMR (400 MHz, Chloroform-d)6.98 (t, J=7.9 Hz, 1H), 6.12 (d, J=9.1 Hz, 1H), 6.06 (d, J=8.8 Hz, 1H),5.96 (s, 1H), 3.89 (s, 2H), 3.78 (s, 3H), 2.92 (m, 2H), 1.22 (m, 1H).¹³C NMR (151 MHz, cdcl₃) 171.68, 148.12, 147.48, 130.20, 105.76, 103.94,99.76, 52.23, 45.66.

HRMS (ES+) calc'd for C₉H₁₃N₂O₂ (M+H) 181.0899 m/z Found. 181.0876.Intermediate vi (20 mg, 0.1 mmol) was dissolved in 1 ml of drynitrobenzene to which the di-bromo intermediate ii (40 mg, 0.11 mmol)and anhydrous DIPEA (46 ul, 0.26 mmol) was added. The solution wasstirred at room temperature for five minutes and anhydrous AlCl₃ (35.6mg, 0.26 mmol) was added accompanied by a fast colour change to deepred). The reaction mixture was diluted with DCM and added directly to anISCO flash column and purified with a gradient of 0-40% DCM/EtOAc over35 min with each solvent containing 0.1% acetic acid. The red solidmono-brominated product intermediate VII was obtained in 27.5% yield (13mg, 0.0275 mmol). ¹H NMR (500 MHz, Chloroform-d) 11.36 (s, 1H), 8.57 (d,J=8.0 Hz, 1H), 8.16 (d, J=7.8 Hz, 1H), 7.80 (d, J=2.5 Hz, 2H), 7.68 (t,J=8.1 Hz, 1H), 7.31 (t, J=8.0 Hz, 1H), 6.75 (d, J=6.6 Hz, 1H), 6.61 (d,J=10.3 Hz, 1H), 6.53 (s, 1H), 4.51 (t, J=5.3 Hz, 1H), 3.96 (d, J=5.4 Hz,2H), 3.82 (s, 3H). ¹³C NMR (151 MHz, DMSO) 179.58, 171.98, 156.75,151.18, 150.04, 149.26, 137.92, 133.39, 132.49, 130.69, 129.89, 128.49,128.35, 124.93, 122.79, 118.82, 117.25, 112.37, 111.72, 107.67, 100.77,52.16, 44.66. HRMS (ES+) calc'd for C₂₃H₁₇BrN₃O₄(M+H) 478.0324 m/z.Found. 478.0311 The mono-brominated intermediate VII (13 mg, 0.0275mmol) was coupled to ABT-1, deprotected, and purified by C18 reversephase chromatography as described above for derivative 1-ABT-1 resultingin the isolation of 4-ABT-1 as a red solid in 46% yield (11 mg, 0.0126mmol). ¹H NMR (500 MHz, DMSO-d₆) 11.71 (s, 1H), 8.91 (t, J=5.4 Hz, 1H),8.76 (d, J=2.7 Hz, 1H), 8.45 (m, 2H), 8.21 (br m, 1H), 8.16 (m, 2H),7.82 (t, J=7.6 Hz, 1H), 7.70 (t, J=7.5 Hz, 1H), 7.14-7.12 (m, 2H), 6.59(m, 2H), 6.49 (br m, 1H), 6.46 (s, 1H), 4.5 (m, 4H), 3.58-3.38 (m, 14H),3.07 (m, 2H), 2.58 (m, 1H), 1.86 (m, 4H), 1.60 (m, 4H). ¹³C NMR (151MHz, DMSO) 175.00, 172.56, 171.44, 165.32, 153.78, 150.42, 148.60,148.51, 146.20, 138.86, 134.92, 133.32, 131.60, 130.27, 129.83, 128.73,127.89, 123.92, 123.82, 122.21, 119.00, 115.41, 110.83, 110.05, 106.16,95.45, 95.17, 70.19, 70.14, 69.93, 68.71, 68.46, 41.42, 40.85, 40.47,36.12, 29.65, 28.66, 27.75. HRMS (ES+) calc'd for C₄₄H₄₉N₈O₁₂ (M+H)881.3470 m/z. Found 881.3536 m/z.

Synthesis of ARM-U2 Derivative 5-ABT-1

Two equivalents of predried DIPEA (452 ul, 2.6 mmol) was added directlyto solid metanilic acid (0.227 g, 1.3 mmol) and the slurry dissolved in3 angstrom pre-dried nitrobenzene (2 ml). To this solution was addeddi-bromo intermediate II (0.1 g, 0.26 mmol) and the solution sonicatedfor 15 min. To this slurry was added anhydrous AlCl₃ (0.182 g, 1.37mmol) and the thick red solution was stirred for 3 hr at roomtemperature (Scheme S5). The reaction mixture was diluted with DCM/MeOH(95:5) and loaded directly onto an ISCO silica column and purified usinga gradient of DCM/MeOH (0-20%) with 1% acetic acid in each solvent. Thecrude mono-sulfonated intermediate VIII (approx. 89 mg, 0.189 mmol, 73%yield) was used directly in the next step. Intermediate VIII (10 mg,0.021 mmol) was coupled to linker ABT-1 as described above for thesynthesis of 1-ABT-1. Crude 5-ABT-1 was purified as described above for1-ABT-1 only with slight modifications to the HPLC conditions nowemploying a 20%-100% gradient over 35 min with 10 mM ammonium formate pH4 buffer/methanol as aqueous/organic phases respectively. ARM-U2derivative 5-ABT-1 was isolated in 35% yield (6.5 mg, 0.00735 mmol) as ared solid. ¹H NMR (500 MHz, DMSO-d₆) 11.74 (s, 1H), 8.90 (t, J=5.5 Hz,1H), 8.76 (d, J=2.7 Hz, 1H), 8.46 (d, J=8.0 Hz, 1H), 8.21-8.12 (m, 2H),8.00 (t, J=4.9 Hz, 1H), 7.83 (t, J=7.5 Hz, 1H), 7.74 (m 1H), 7.76-7.69(m, 1H) 7.51 (d, J=6.4 Hz, 1H), 7.48 (t, J=7.5 Hz, 1H), 7.44 (d, J=7.8Hz, 1H), 7.13 (d, J=9.7 Hz, 1H), 6.40 (s, 1H), 4.48 (br s, 4H),3.62-3.33 (m, 14H), 3.09 (q, J=6.7, 6.3 Hz, 2H), 2.54 (m, 1H), 1.85 (m,4H), 1.61 (m, 4H). ¹³C NMR (151 MHz, DMSO) 175.42, 172.37, 154.20,153.35, 150.04, 148.57, 148.51, 146.01, 137.87, 134.89, 133.27, 131.82,130.27, 129.92, 129.79, 128.94, 127.98, 123.94, 123.67, 122.91, 122.32,120.61, 118.99, 115.42, 95.83, 94.82, 70.14, 69.92, 68.69, 68.48, 55.38,49.04, 41.41, 36.12, 29.57, 28.66, 27.87. HRMS (ES+) calc'd forC₄₂H₄₆N₇O₁₃S (M+H) 888.2874 m/z. Found 888.2927.

Synthesis of ARM-U2 Derivative 6-ABT-1

To a flame-dried flask under argon was added the di-bromo intermediateII (0.020 g, 0.052 mmol) Li₂CO₃ (0.029 g, 0.4 mmol),aniline-2,4-disulfonic acid (0.100 g, 0.4 mmol) and Cu(OAc)₂ (0.0005 g,0.0027 mmol). The flask was sealed and flushed with argon followed bythe addition of 4 ml anhydrous DMF via syringe. The solution was stirredfor 5 min under argon at room temperature and then heated to 70° C. for24-36 hr shielded from room light. After reaction completion monitoredby LCMS and reverse-phase TLC, the reaction mixture was precipitated outof a 12:1 (w,v) solution of diethylether/DMF at −20° C. and the isolatedred precipitate dissolved in water and loaded onto a reverse phase C18column. Crude intermediate VIIII was purified by reverse phase C18-ISCOchromatography in a dark room (0-80% H₂O/MeOH 0.1% formic acid/30 min).The isolated pinkish fractions were pooled and lyophilized giving riseto pure intermediate VIIII isolated in 55% yield (15.7 mg, 0.028 mmol).¹H NMR (500 MHz, DMSO-d₆) 11.72 (s, 1H), 8.46 (d, J=7.8 Hz, 1H), 8.20(d, J=7.8 Hz, 1H), 8.14 (s, 1H), 7.92 (t, J=7.6 Hz, 1H), 7.79 (t, J=7.7Hz, 1H), 7.73 (s, 1H), 7.67 (d, J=8.0 Hz, 1H), 7.51 (d, J=8.1 Hz, 1H).¹³C NMR (151 MHz, DMSO) 179.21, 157.13, 151.39, 147.50, 145.95, 140.55,134.54, 133.32, 132.78, 130.04, 129.02, 128.76, 127.35, 125.95, 125.00,124.21, 122.72, 117.87, 117.62, 102.27. HRMS (ES+) calc'd forC₂₀H₁₂BrN₂O₈S₂ (M+H) 549.9140 m/z. Found 549.9127. Intermediate VIIII (8mg, 0.014 mmol) was dissolved in anhydrous DMF (2 ml) to which anhydrousDIPEA (12.2 ul, 0.07 mmol) and linker ABT-1 (0.014 g, 0.028 mmol) wereadded and the solution allowed to stir for 5 hr at 70° C. The reactionmixture was concentrated under a stream of nitrogen, filtered andpurified directly by C18 HPLC chromatography (20-100% H₂O/MeOH, ammoniumformate buffer pH4/30 min). The obtained fractions were pooled andlyophilized resulting in pure 6-ABT-1 isolated in 42% yield as theammonium formate salt (5.7 mg, 0.0058 mmol) as an orange-reddish solid(accurate yield determination confirmed by UV using an extinctioncoefficient of 17,000 for the DNP unit). ¹H NMR (500 MHz, DMSO-d₆) 11.71(s, 1H), 8.93 (t, J=5.4 Hz, 1H), 8.79 (d, J=2.6 Hz, 1H), 8.45 (d, J=8.0Hz, 1H), 8.24 (brs, 1H), 8.21 (dd, J=9.6, 2.6 Hz, 1H), 8.13 (m, 2H),7.86 (t, J=5.4 Hz, 1H), 7.81 (t, J=7.5 Hz, 1H), 7.69 (t, J=7.6 Hz, 1H),7.62 (d, J=8.1 Hz, 1H), 7.55 (d, J=8.2 Hz, 1H), 7.19 (d, J=9.7 Hz, 1H),6.38 (s, 1H), 4.64 (brs, 2H), 4.18 (brs, 2H), 3.58-3.37 (m, 14H), 3.10(m, 2H), 2.53 (m, 1H), 1.92-1.80 (m, 4H), 1.65-1.54 (m, 4H). ¹³C NMR(126 MHz, DMSO) 175.29, 172.59, 154.22, 152.01, 148.64, 145.44, 144.72,140.10, 135.64, 135.00, 133.70, 131.70, 130.39, 129.93, 128.88, 128.30,126.89, 126.27, 124.10, 123.99, 123.68, 122.10, 119.46, 115.58, 97.18,96.81, 70.15, 69.95, 68.74, 68.45, 52.24, 42.73, 41.46, 36.19, 29.66,28.67, 27.97, 24.17. HRMS (ES+) calc'd for C₄₂H₄₆N₇O₁₆S₂(M+H) 968.2442m/z. Found 968.2420.

Synthesis of ARM-U2 Derivative 6-ABT-2

ARM-U2 derivate 6-ABT-2 was synthesized and purified as described abovefor 6-ABT-1 only intermediate VIIII (8 mg, 0.014 mmol) was coupled tolinker ABT-2 (9 mg, 0.014 mmol) as opposed to ABT-1 resulting in theisolation of 6-ABT-2 in 35% yield as the ammonium formate salt (0.0049mmol, 5.5 mg). ¹H NMR (600 MHz, DMSO-d₆) 11.71 (s, 1H), 8.81 (m, 2H),8.45 (d, J=8.0 Hz, 1H), 8.21 (dd, J=9.6, 2.6 Hz, 1H) 8.13 (m, 2H), 8.00(t, J=5.5 Hz, 1H), 7.81 (t, J=7.5 Hz, 1H), 7.70 (t, J=7.6 Hz, 1H), 7.62(d, J=9.6 Hz, 1H), 7.55 (d, J=8.2 Hz, 1H), 7.26 (d, J=9.6 Hz, 1H), 6.38(s, 1H), 4.63 (brs, 2H), 4.17 (brs, 2H), 3.73-3.08 (m, 32H), 2.58 (m,1H), 1.87 (dd, J=30.1, 11.0 Hz, 2H), 1.74-1.66 (m, 1H), 1.59 (m, 1H).¹³C NMR (151 MHz, DMSO) 175.29, 172.90, 164.96, 154.22, 151.91, 148.75,148.59, 145.39, 144.55, 139.96, 135.65, 135.19, 133.65, 131.75, 130.28,129.92, 128.94, 128.28, 126.93, 126.20, 124.07, 123.92, 123.72, 122.14,119.43, 116.14, 97.18, 96.78, 70.17, 69.38, 68.62, 55.39, 43.05, 40.79,38.87, 28.10, 24.18. HRMS (ES+) calc'd for C₄₈H₅₈N₇O₂₀S₂(M+H) 1116.3178m/z. Found 1116.3281.

Synthesis of ARM-U2 Derivative 6-ABT-3

ARM-U2 derivative 6-ABT-3 was synthesized and purified exactly asdescribed above for ARM-U2 6-ABT-1 only intermediate VIIII (8 mg, 0.014mmol) was coupled to linker ABT-3 (28 mg, 0.025 mmol) as opposed toABT-1 resulting in the isolation of 6-ABT-3 in 27% yield as the ammoniumformate salt (6 mg, 0.0038 mmol). ¹H NMR (600 MHz, DMSO-d₆) 11.71 (s,1H), 8.84 (d, J=2.7 Hz, 1H), 8.46 (d, J=7.9 Hz, 1H), 8.31 (br s, 2H),8.24 (dd, J=9.6, 2.7 Hz, 1H), 8.14 (d, J=7.8 Hz, 1H), 8.13 (d, J=2.1 Hz,1H), 8.01 (t, J=5.6 Hz, 1H), 7.82 (m, 2H), 7.71 (t, J=7.6 Hz, 1H), 7.62(d, J=8.2 Hz, 1H), 7.56 (d, J=8.2 Hz, 1H), 7.28 (d, J=9.7 Hz, 1H), 6.39(s, 1H), 4.63 (br s, 2H), 4.19 (br s, 2H), 3.69-3.17 (m, 64H), 2.56 (s,1H), 2.03 (t, J=7.5 Hz, 4H), 1.91-1.83 (m, 2H), 1.73-1.64 (m, 3H),1.63-1.59 (m, 1H). ¹³C NMR (151 MHz, DMSO) 175.32, 172.89, 172.22,165.01, 154.23, 151.97, 148.79, 148.63, 145.42, 144.64, 140.05, 135.65,135.27, 133.70, 131.73, 130.30, 130.04, 128.91, 128.30, 126.90, 126.23,124.12, 123.95, 123.71, 122.13, 119.47, 116.14, 97.24, 96.81, 70.23,69.55, 68.68, 52.14, 49.17, 43.09, 42.53, 38.86, 35.10, 28.07, 24.18,21.93. HRMS (ES+) calc'd for C₆₉H₉₈N₉O₂₉S₂(M+H) 1580.5912 m/z. Found1580.6155.

Synthesis DNP Substituted Analog 6-ABT-4

Synthesis an analog of derivative 6-ABT-1 where the DNP moiety issubstituted by pyrimidine expected and later shown experimentally tohave no affinity for anti-DNP antibodies was initiated by coupling of2-bromopyrimidine to the diamine3,3′-((oxybis(ethane-2,1-diyl))bis(oxy))bis(propan-1-amine) usingHartwig-Buchwald Pd(0) coupling conditions however this was unsuccessfullikely due to deactivating chelation of the amine ligand to palladium.Alternative Ullman-type conditions were employed by dissolving thediamine (100 mg, 0.45 mmol) and 2-bromo-pyrimidine (57 mg, 0.36 mmol) in2 ml deI water open to atmosphere followed by the addition of coppermetal (1.4 mg, 0.023 mmol). Upon heating for 1 hr at 100° C. thesolution turned a bluish-green and the reaction was quenched using 1MHCL. The solution was concentrated and purified by ISCO C18chromatography to yield the pyrimidine-monoamine linker intermediate Xin 80% yield as a slightly yellowish oil (86 mg, 0.29 mmol) ¹H NMR (500MHz, DMSO-d₆) 8.42 (s, 2H), 8.24 (d, J=4.7 Hz, 2H), 7.08 (t, J=5.4 Hz,1H), 6.53 (t, J=4.7 Hz, 1H), 3.54-3.42 (m, 10H), 3.29 (q, J=6.6 Hz, 4H),2.77 (t, J=7.2 Hz, 2H), 1.73 (dp, J=13.7, 6.5 Hz, 4H). HRMS (ES+) calc'dfor C₁₄H₂₇N₄O₃ (M+H) 299.2005. Found 299.2011. Intermediate X (86 mg,0.29 mmol) was coupled to Boc-L-nipecotic acid and deprotected asdescribed above for linker ABT-1 to yield ABT-4 in quantitative yield(118 mg, 0.29 mmol) and used directly in the next step. The di-bromointermediate VIIII (8 mg, 0.014 mmol) was coupled to linker ABT-4 (11.4mg, 0.028 mmol) and purified exactly as described above for 6-ABT-1resulting in the isolation of 6-ABT-4 in 43% yield (5.2 mg, 0.006 mmol)¹H NMR (500 MHz, DMSO-d₆) 11.70 (s, 1H), 8.43 (d, J=8.1 Hz, 1H), 8.26(d, J=4.6 Hz, 2H), 8.14 (d, J=12.6 Hz, 2H), 7.86 (t, J=5.4 Hz, 1H), 7.81(t, J=7.5 Hz, 1H), 7.69 (t, J=7.6 Hz, 1H), 7.62 (d, J=9.9 Hz, 1H), 7.55(d, J=8.2 Hz, 1H), 7.07 (brs, 1H) 6.55 (t, J=4.8 Hz, 1H), 6.37 (s, 1H),4.62 (brs, 2H), 4.18 (brs, 2H), 3.51-3.35 (m, 10H), 3.26 (m, 4H),3.16-3.04 (m, 2H), 1.92-1.78 (m, 1H), 1.71 (t, J=10.0 Hz, 4H), 1.65 (m,4H). ¹³C NMR (151 MHz, DMSO) 175.30, 172.60, 163.47, 158.09, 154.26,152.01, 148.65, 145.47, 144.63, 139.97, 135.67, 133.67, 131.75, 128.92,128.28, 126.95, 126.26, 124.12, 123.77, 122.16, 119.46, 110.11, 97.17,96.80, 70.17, 69.94, 68.64, 68.42, 52.22, 49.16, 42.70, 38.49, 36.16,29.66, 29.42, 28.00, 24.17. HRMS (ES+) calc'd for C₄₀H₄₆N₇O₁₂S₂(M+H)880.2568 m/z. Found 880.2538.

Results and Discussion Computation.

Inspiration for the development of the target binding terminus (TBT) ofARM-U2 came from studies of literature reported anthroquinone derivedcompound IPR-803 (T. Mani, S. Meroueh et al) with triple digit nMaffinity for uPAR. Computational modeling studies were conducted toassess the optimal location for installing the antibody recruiting motifonto the anthroquinone when complexed to uPAR (FIG. 2). ComputationaluPAR docking studies suggested that functionalization of the IPR-803core with a dinitrophenyl-trioxa-1,13-tridecanediamine antibody bindingterminus (ABT) at position 2 of ring C (ARM-U2 1-ABT-1, FIG. 4,Scheme 1) would allow for the antibody recruiting motif to remainsolvent exposed available to bind endogenous anti-DNP antibodies in vivoand not interfere with uPAR binding affinity. Computational dockingstudies were also conducted to guide the development of higher affinitybinding ARM-U2 derivatives by docking compounds into the uPA bindingsite of uPAR where IPR-803 interactions with “hot spot” residues areproposed to occur (T. Mani, S. Meroueh et al).

The above process was repeated for derivatives differing in the display,density, and type of negative charge about the aniline moietysubstituted at ring C of IPR-803 (FIG. 4, Scheme 1, FIG. 8, Table 1).Computational docking of designed ARM-U2 derivatives with an additionalcarboxyl moiety substituted at the aniline ring ARM-U2 2-ABT-1 or asingle meta carboxyl to meta-sulfonate substitution ARM-U2 5-ABT-1predicted no increase in uPAR binding affinity (See FIG. 4, Scheme 1,FIG. 8, Table 1). However derivatives with the carboxyl moiety extendedfurther from the aniline ring (3-ABT-1 and 4-ABT-1) and substitution ofa single meta-sulfonate for two sulfonates at the ortho and paraposition 6-ABT-1 were predicted to experience significant gains inbinding energy (See FIG. 4, Scheme 1, FIG. 8, Table 1). These increasesare predicted by computation to be due to a gain in H-bonding capabilitywith Arg-53 and Lys-50.

The results of the computational studies supported the chemicalsynthesis of ARM-U2 derivatives with the ABT appended to the core ofIPR-803 at position 2 of ring C for further in vitro uPAR bindingstudies. The results of the docking experiments also suggested thatuPAR-binding affinity may be tunable by optimizing compound interactionswith hot-spot basic residues in the uPA-binding pocket (1-ABT-1 vs4-ABT-1 and 6-ABT-1, FIG. 8, Table 1). The bi-functional TBT and ABTcontaining ARM-U2 derivatives that were docked to uPAR were thenchemically synthesized to evaluate in subsequent in-vitro uPAR bindingassays

Longer ABTs consisting of longer PEG-derived building blocks weresynthesized chemically and incorporated into additional ARM-U2derivatives in attempts to optimize antibody binding affinity.

Competitive ELISA uPAR Binding Assays.

The affinity of ARM-U2 derivatives differing in both the length of theDNP-linker substituted at position 2 of ring C and the display ofnegative charge density (FIG. 8, Table 1) for uPAR were assayed using acompetitive ELISA binding assay. The assay involves the immobilizationof urokinase, a natural high affinity ligand for uPAR followed by theaddition of a fixed concentration of recombinant uPAR protein andincreasing concentrations of ARM-U2 which competes with urokinase forbinding to the uPA binding site of uPAR. Following several washingsteps, the amount of uPAR bound is quantified by a biotinylatedanti-uPAR antibody and avidin-HRP and can be fit to a competitivebinding model allowing for the extraction of the uPAR/ARM-U2 K_(d).

All ARM-U2 derivatives were observed to bind to uPAR selectively at theuPA binding site with single to triple digit nM affinity. These resultsconfirmed the previous computational predictions that installing the ABTat position 2 of ring C of the anthroquinone core enabled for ARM-U2parent derivative 1-ABT-1 to retain the expected triple digit nanomolarbinding affinity for uPAR (as reported in the literature for parentcompound IP-803 (Table 1, FIG. 8). Longer linker-containing derivatives(ABT-2 and ABT-3) showed an increased binding affinity compared to theirshorter trioxa-1,13-tridecanediamine linker-containing counterpart(ABT-1) however this is likely attributed to additional interactions ata distal arene binding site on uPAR.

The uPAR-binding affinities calculated for compounds 3-ABT-1 and 4-ABT-1which have the carboxylic acid extended further from the aniline moietyand predicted to bind uPAR with higher affinity by computation wereshown to have a modest increase in affinity relative to parent ARM-U2derivative 1-ABT-1. The di-acid derivative 2-ABT-1 and mono-sulfonate5-ABT-1 also showed no increase in uPAR-binding affinity relative toparent ARM-U2 1-ABT-1. This observation suggests that the interactionswith uPAR are not only electrostatic in nature but could be highlydirectional. Derivative 6-ABT-1 however, containing an ortho and parasulfonate showed a significant increase in affinity relative to 1-ABT-1.The increase in binding affinity observed for 6-ABT-1 is likely theresult of its ability to make specific H-bonds with Arg-53 and Lys 50 aspredicted by docking studies in contrast to 1-ABT-1. This is furthersupported by the fact that 6-ABT-1 binds uPAR with significantly higheraffinity than double carboxylate derivative 2-ABT-1 which cannot beexplained by long range acting electrostatic interactions alone but canbe explained considering the known stronger H-bonding and higherenthalpy of interaction that occurs between guanidinium functionalitiesand sulfonate groups compared to carboxylate groups.

From these in vitro binding studies, we can conclude that computationaldocking studies accurately predicted compounds with increasing uPARbinding affinity in addition to the correct location to substitute withthe ABT to avoid disruption of uPAR binding. We can also conclude thatan anthroquinone derived small molecule is an effective substitute forthe large uPA target binding motif on ARM-U and that ARM-U2 canpotentially recruit antibodies to the surface of uPAR-expressing cancercells inducing cellular phagocytosis and cytotoxicity at single totriple digit nanomolar concentrations.

uPAR/6-ABT-1 Crystallization Studies

To reinforce the results of our competitive binding assays in that6-ABT-1 binds to the uPA binding site of uPAR via the uPA binding siteand support our hypothesis that the increase in affinity is due tospecific interactions with cationic hotspot residues within the bindingpocket of uPAR, we investigated the binding of 6-ABT-1 to crystallizeduPAR protein in collaboration with Dr M. Huang who soaked the compoundinto pre-crystallized uPAR. We observed that 6-ABT-1 binds to uPAR atthe uPA binding interface with the antibody recruiting motif solventexposed while making a specific contact with Arg-53 previously reportedto be a hotspot residue engaged by urokinase (FIG. 2). This result alsosuggests a potential dual mode of action as an anti-cancer therapeuticacting as an antagonist of the oncogenic uPA-uPAR interaction inaddition to acting as an antibody-recruiting molecule against uPAR.

Antibody Recruiting Cellular Assays

Next, we tested the ability of ARM-U2 derivatives to recruit anti-DNPantibodies to the surface of A-172 and B-16 cancer cells over-expressinguPAR. These assays involved the incubation of ARM-U2 derivatives withtarget cancer cells followed by the addition of biotinylated anti-DNPantibodies and streptavidin-AlexaFluor conjugates. After several washingsteps, the amount of bifunctional ARM-U2 compound bound simultaneouslyto both the cell surface and anti-DNP antibodies was detected using flowcytometry by observing shifts in FL-2 and FL-3 fluorescence. Theseexperiments were also conducted on both B16+uPAR cell lines and B16−uPARcell lines in addition to studies on A172 cells in the presence andabsence of exogenously added competitor uPAR to assess selective bindingto the cell surface via cell surface uPAR.

Derivatives 1 to 6-ABT-1, and longer ABT containing variants 1-ABT-2/3and 6-ABT-2/3 all demonstrated the ability to survive multiple washingsteps and simultaneously bind to both anti-DNP antibodies and cellsurface uPAR on A172 cells with antibody recruiting capability decliningwith increased linker length. High affinity uPAR binding derivative6-ABT-1 recruited antibodies significantly better than the other ABT-1derivatives on A172 cells and antibody recruitment could be disrupted byadding exogenous competitor uPAR. ARM-U2 6-ABT-1 also recruited anti-DNPantibodies to the surface of B-16+uPAR cells but failed to do so withB-16−uPAR cells (FIG. 9A, B).

From these results we can conclude the following:

-   -   1. The location on the anthroquinone chosen for linker        substitution and length of ABT-1 appears to be effective for        engaging in interactions with both cell surface uPAR and        anti-DNP antibodies confirming its solvent exposed environment        accordance with predictions made from computation;    -   2. The observed trend of decreasing antibody-recruiting        capability with increasing linker length further supports the        argument for an interaction between the antibody recruiting        motif and a distal arene binding site on uPAR becoming less        available for anti-DNP antibody interactions while engaged in        binding uPAR contributing to the higher uPAR binding affinity        observed. (FIG. 8, Table 1)    -   3. The ability of exogenously added uPAR to disrupt the antibody        recruiting capability of 6-ABT-1 supports the selective binding        of 6-ABT-1 to cell surface uPAR in the presence of the complex        cellular meilleur;    -   4. This fact is further supported by the selective antibody        recruiting mediated by 6-ABT-1 to B16+uPAR cell lines and not        B16−uPAR (FIG. 9A,B);    -   5. The ability of all the derivatives to survive the multiple        washing steps required in the antibody-recruiting flow cytometry        assay combined with the knowledge that 6-ABT-1 recruits anti-DNP        antibodies much more effectively than the other derivatives        despite identical antibody-recruiting motifs, supports its        significantly higher uPAR binding affinity with an expected        dissociation half life on the order of 1-2 min enabling for more        compound to remain bound to the cells, recruit antibodies and        survive washing steps.

ARM-U2 Mediated Antibody Dependent Cellular Phagocytosis (ADCP)

ADCP experiments carried out via a dual channel flow cytometry assay andwere performed by measuring the % phagocytosis of A172 humanglioblastoma cells by IFN-gamma primed U937 macrophage effector cellseach stained with FL-1 and FL-4 dyes respectively giving rise to doublepositive FL-1 and FL-4 fluorescence shifts accompanying phagocytosis.These assays were performed in the presence and absence of exogenouslyadded anti-DNP antibodies and various concentrations of each ARM-U2derivative. Control experiments were also performed to confirm thatcellular phagocytosis is occurring and is dependent on ARM-U2 bindingcell surface uPAR by

-   -   1. Performing out competitions with exogenous uPAR,    -   2. Performing out-competitions with 6-ABT-4 which should bind        uPAR but not anti-DNP antibodies    -   3. Performing ADCP at 4 degrees celcius where phagocytosis        cannot occur and    -   4. Amnis microscopic analysis of double positives identified in        the ADCP assay to undergo 6-ABT-1 dependent phagocytosis.

ADCP experiments were also performed on B16+uPAR and −uPAR cell lines asan isogenic control to assess selectivity for cell surface uPAR. All ofthe ARM-U2 derivatives tested were observed to induce U937monocyte-mediated phagocytosis of A172 glioblastoma cells overexpressinguPAR at ARM-U2 concentrations spanning single to triple digit nanomolarconcentrations. Increasing linker length was observed to result in adecrease in ADCP efficacy when comparing 1-ABT-1 vs 1-ABT-2/3 as well as6-ABT-1 vs 6-ABT-2/3. The longer linker ABT-2 and ABT-3 functionalizedARM-U2 derivatives with lower antibody recruiting capability than theirrespective ABT-1 functionalized counterparts, showed lower ADCP efficacyin accordance with our hypothesis that longer antibody recruiting motifsare capable of binding to a distal site on uPAR resulting in anincreased binding affinity however less available for interactions withanti-DNP antibodies. Increasing the negative charge density (2-ABT-1)and further extending the carboxylic acid moiety (3-ABT-1, 4-ABT-1)showed no increase in efficacy or potency in ADCP compared to compounds1-ABT-1 in accordance with the similar binding affinities calculated foruPAR binding and similar antibody recruiting capability observed by flowcytometry.

The lack of effect of increasing the negative charge density at theaniline substituted at ring C (2-ABT-1 vs 1-ABT-1) reinforces thehypothesis that ARM-U2 interactions with the basic residues in the uPAbinding pocket are not purely electrostatic in nature and are likelymediated through specific H-bonding. Compound 5-ABT-1 containing onemeta-substituted sulfonate showed no increase in % phagocytosis comparedto the mono carboxylic acid derivative 1-ABT-1 in accordance with itsrelatively similar uPAR binding affinity and antibody recruitingcapability. The fact that the % phagocytosis does not changesignificantly with the di-acid or mono-sulfonate derivatives (2-ABT-1and 5-ABT-1) further argues against phagocytosis occurring as a resultof non-specific hydrophobic or electrostatic interactions with the cellsurface, since the additional negative charge density on these compoundswould be expected to cause an increase in efficacy if these compoundsbound to basic cell surface proteins and a decrease in efficacy if theyinteracted hydrophobically with the cell surface. Doublesulfonate-substituted compound 6-ABT-1 with sulfonates at the ortho andpara positions however showed a significant increase in potency andefficacy compared to the mono-acid derivative 1-ABT-1 as predicted bycomputation.

As ARM-U2 derivative 6-ABT-1 bound to uPAR with the highest affinity(FIG. 8, Table 1), and was shown to recruit anti-DNP antibodies mosteffectively (FIG. 9A,B), it follows that this derivative shows thehighest efficacy and potency of all the ARM-U2 derivatives tested inADCP (FIG. 9C). The sharp increase in % phagocytosis observed for6-ABT-1 between 10 and 50 nM corresponds well with the calculateddissociation constant of 12 nM and supports a specific uPAR-dependentphagocytic response (FIG. 9C). The high selectivity of 6-ABT-1 and thedependence of its efficacy in ADCP on cell surface uPAR is demonstratedby the ability to disrupt phagocytosis by the addition of exogenous uPARwhich competes with cell surface uPAR for binding 6-ABT-1 (FIG. 9C).This is further demonstrated by the ability to disrupt phagocytosis bythe addition of 6-ABT-4 which can bind uPAR but not anti-DNP antibodiesconfirming that the efficacy of 6-ABT-1 is dependent on a specific uPARbinding interaction and not a non-specific interaction with the cellsurface which would be unsaturatable and unable to be competed off bythe addition of 6-ABT-4 (FIG. 9C). The bell shaped appearance of thephagocytosis efficacy dependence on 6-ABT-1 concentration is typical ofthe well documented prozone effect and is a further indication of6-ABT-1 selectivity for cell surface uPAR (FIG. 9C). The downward slopeon the right hand side of the bell shaped curve arises at highconcentrations of 6-ABT-1 (>100 nM) due to a competition for limitedanti-DNP antibody between cell surface bound and unbound 6-ABT-1 andwould not occur of 6-ABT-1 bound to the cell surface non-specificallyand therefore in a non-saturatable manner.

The inventors hypothesize that the increased efficacy and selectivity of6-ABT-1 relative to the other ARM-U2 derivatives studies is primarilydue to its significantly enhanced affinity for uPAR and is also in partdue to its high negative charge density which increases themonodispersion of the compound in aqueous media while decreasing theamount of non-specific binding that can potentially occur in thenegatively charged environment of the cell surface. The efficacy of6-ABT-1 is confirmed to be cellular phagocytosis by the demonstrationthat relatively few double positives are observed in the ADCP assay at 4degrees celcius and by Amnis analysis of ADCP samples of 6-ABT-1 at 37degrees celcius which illuminates the microscopic engulfment of stainedA172 target cells by stained u937 monocytes (FIG. 9D). The significantincrease in ADCP efficacy and potency observed for thedisulfonate-containing ARM-U2 derivative 6-ABT-1 is again supportive ofstronger interactions with basic residues in the uPA-binding site ofuPAR, including Arg-53 supported by crystallography in addition tolysine 50 and glutamic acid 68 implicated by computational dockingexperiments.

Taken together, the inventors have shown that computationally guidedARM-U2 design enabled for significant gains in uPAR binding affinity tobe achieved through the facile synthetic tuning of negative chargedisplay and density about the functionalized aniline substituted atposition 4 of ring C of the anthroquinone scaffold, (FIG. 4, Scheme 1,FIG. 8, Table 1).

ARM-U2 Mediated Antibody Dependent Monocytic u937 Cell Release ofInflammatory Cytokines

Derivative 6-ABT-1 was shown through a multi-cytokine immobilized ELISAto stimulate a higher release of IL-8 by u937 cells through therecruitment of anti-DNP antibodies to the surface of A-172 cells whichsubsequently stimulate u937 monocytes presumably through Fc gammareceptor clustering. IL-8 is primarily released by monocytes andmacrophages early in the innate immune response upon recognition of aforeign antigen which initiates the immune cascade.

ARM-U2 Mediated Inhibition of Tumor Progression in an Allograft MouseB16 Tumor Model

IP injections of 6-ABT-1 over the course of 17 days was shown tosignificantly halt tumor progression relative to the PBS control inlaboratory test animals (mice). After 17 days of treatment with 6-ABT-1or PBS, un-treated mice were observed to possess significant tumorburden reaching mean tumor volumes of 1000 mm³ at which point they weresacrificed as dictated by standard ethics protocols where 6-ABT-1treated mice in two separate cohorts given two different dosingconcentrations possessed tumors with an average volume of approximately200 mm³ overlapping with that observed for the doxorubicin treatedcohort (FIG. 10).

To further assay effector cell activation by ARM-U2, we investigated therelease of pro-inflammatory cytokines by U937 monocytes in the contextof ADCP assays. Thus, cellular supernatants were isolated from ADCPassay conditions and evaluated using a multi-inflammatory cytokine ELISAformat. The results are shown in FIG. 11. Higher levels of IL-8secretion were observed in assays containing 50 nM or 1 μM ARM-U2compared to control samples lacking ARM-U2 (FIG. 11B).

The inventors also examined the efficacy of ARM-U2 in a B16−uPAR mouseallograft model developed in our laboratory. Mice immunized to produceanti-DNP IgG antibodies containing tumors expressing human uPAR,received daily treatment via intraperitoneal (IP) injection and weremonitored over the course of 42 days. Mice treated with ARM-U2 (20 mpkor 100 mpk) showed a significant decrease in the rate of tumor growthrelative to those injected with PBS only, corresponding to a calculatedtumor growth inhibition (TGI) of approximately 90% (See FIG. 12A). ThisTGI level was comparable to mice treated with doxorubicin (Dox, 1 mpk),an anti-mitotic chemotherapeutic agent often used as standard-of-care intreating metastatic malignancies. For animals treated with eitherdoxorubicin or ARM-U2, tumor regression persisted almost 14 daysfollowing treatment cessation at which point animals with excessivetumor burden were sacked. Interestingly, mice treated with the 100 mpkdose of ARM-U2 showed a moderate decrease in efficacy especially withinthe first 22 days of treatment compared to the mice dosed with 20 mpk ofARM-U2 (FIG. 12A). This reduction in efficacy observed in vivo in the100 mpk dosing regimen is consistent with a “prozone” mechanism ofaction as observed in cellular ADCP and ADCC assays, although othermechanisms might also be involved. Mean survivals of 27 and 30 days wereobserved for mice treated with doxorubicin and ARM-U2 respectivelyrelative to a mean survival of 17.5 days for the PBS control (FIG. 12B).Tumor regression was even more pronounced in the combination treated armof the study with a mean survival of 37 days. Remarkably, mice treatedwith ARM-U2 at both 20 mpk and even 100 mpk did not lose weight incontrast to that observed accompanying treatment with doxorubicin.Although lack of weight loss is not a definitive indicator of compoundsafety, these results represent a positive indication that ARM-U2 mightpossess an improved side-effect profile compared to doxorubicin (FIG.12C).

Further Studies ARM-U2 Fluorescence and ELISA Binding Studies

Selected ARM-U2 derivatives were assayed for their ability to bindrecombinant uPAR using a fluorescence quenching assay. The compoundsstored at −20° C. as DMSO stocks were diluted into PBS aliquots at afixed 50 nM concentration. Increasing concentrations of recombinant uPARwere mixed with each compound containing aliquot and the fluorescence ofthe solution measured following 1 h equilibration times. Increasingconcentrations of uPAR resulted in saturatable quenching of intrinsicARM-U2 fluorescence generating a binding isotherm that could be fit byEquation S1 to extract the equilibrium dissociation constant for ARM-U2binding to uPAR. To confirm that the observation of uPAR dependentfluorescence quenching was indeed due to specific binding to uPAR and tovalidate ARM-U2 binding to uPAR specifically via the uPA binding site, adirect binding ELISA assay was developed. In this assay, clear 96-wellhigh-binding plates from CoStar, were loaded with 100 μl/well of 50 nMrecombinant uPAR 807-UK-100-CF from R&D systems prepared from a 100μg/ml (approx. 2.1 μM) stock of uPAR in PBS and left overnight at roomtemperature to equilibrate. Following equilibration, the plate waswashed with wash buffer 1× (400 μl/well of 0.05% Tween/PBS), blotted dryand blocked for 1 hr with 300 ul/well of 2% BSA/PBS. Following blocking,the plate was washed with 400 ul of the wash buffer and blotted dry.Dilutions of each ARM-U2 derivative from 100× stocks in DMSO were madeinto 100 μl/well of 1% BSA/PBS and left to equilibrate with immobilizeduPAR for 2 h at RT. Following the binding step, the plate was washed1×200 μl and 1×400 μl of wash buffer and blotted dry. A solution ofanti-DNP rabbit IgG KLH biotinylated antibody was prepared in 1% BSA/PBSfrom a 10,000× commercially available stock of 2 mg/ml in 500 μl (LifeTechnologies) and added in a volume of 100 μl/well, left to equilibratefor 1 h at RT. To assess selectivity of binding of ARM-U2 to the uPAbinding site of uPAR, 100 nM competitor uPA-ATF (Innovative Research),the amino terminal fragment of uPA which retains the complete bindingcapability of urokinase for uPAR, was added during the biotinylatedanti-DNP antibody incubation step. Following incubation with theantibody, the plate was washed 1×200 μl 1×400 μl of wash buffer andblotted dry. A solution of avidin-HRP was prepared by diluting 20 μl ofstock solution (e-bioscience) into 10 ml of 1% BSA/PBS solution and 100μl added to each well and incubated for 20 min at RT. Following 3×400 ulwashes and blotting, 100 ul of HRP substrate solution (TMB-substrate,Thermo) was added and the wells allowed to develop until a cleardifference between negative control (no uPAR loaded) and wellscontaining ARM-U2 could be observed by differences in the emergence ofblue color at which time the development was halted by the addition of50 μl 4N sulfuric acid. The absorbance at 450 nm was measured using anin-house Perkin-Elmer fluorescence plate reader.

All ARM-U2 derivatives were assessed for their ability to bindselectively to recombinant uPAR in vitro using a competitive ELISAbinding assay. DMSO stock solutions of each ARM-U2 derivative stored at−20° C. were titrated into wells pre-coated with human urokinase(isolated from human urine, ProSpec 1 mg/ml deI water 18.5 μM) in thepresence of uPAR. Selective binding to uPAR by ARM-U2 at the uPA bindingsite competes with uPAR-uPA binding which is required for a positive UV450 nm absorbance signal. Increasing concentrations of ARM-U2 displaceincreasing amounts of uPAR from uPA which is detected byanti-uPAR/biotin labeled antibodies followed by avidin-HRP. Thesigmoidal binding isotherm of absorbance at 450 nm vs log ARM-U2concentration can then by fit by non-linear regression analysis toobtain the IC₅₀ for ARM-U2/uPAR binding (Equation S2) which can then betranslated into a K_(I) for ARM-U2/uPAR binding using a competitivebinding model adapted from the Cheng-Prusoff equation relating K_(I) toIC₅₀ (Equation S3). The ELISA experiments were performed as followed: ToCorning high binding 96 well flat bottom plates was added 100 μl/well of100 nM urokinase in sterile PBS. The plates were sealed and left toincubate at 4° C. overnight. The plates were then washed 1× with 200 μlwash buffer (PBS+0.05% Tween-20) and 1× with 400 μl wash buffer. Theplates were blotted on paper towel and blocked for 1 h shaking at roomtemp with 300 μl of a solution of thermo superblock in PBS per well.Molecule dilutions into solutions of a fixed concentration (10 nM) ofuPAR were prepared in 0.025% triton-X/PBS on a separate low bindingplate during the blocking hour. The plates were again washed followed bythe addition of 100 μl of the ARM-U2/uPAR solution with 0.1% final DMSOconcentration. The solutions were mixed by multichannel pipette, sealed,and incubated for 2 h with shaking at room temperature. The plates werethen washed 1× with 200 μl wash buffer (PBS+0.05% Tween-20) and 2× with400 μl wash buffer. The plates were blotted on a paper towel followed bythe addition of 100 μl/well of a solution of biotinylated anti-uPARantibody in 1% BSA/PBS. (R&D Systems, 3.3 μl taken from 50 μg/250μl-1000× stock) and incubation with shaking for 1 h at room temp. Theplates were washed 1× with 200 μl wash buffer and 2× with 400 μl washbuffer and blotted on a paper towel. 100 μl of avidin-HRP (stock −500×from e-bioscience) were added per well, incubated 20 min with shaking atroom temp followed by washing 1× with 200 μl wash buffer and 3× with 400μl wash buffer. The plates were blotted dry with paper towel and 100 μlof TMB substrate was added to each well. The emergence of blue color wasmonitored and the reaction quenched with the addition of 50 μl of 2Msulfuric acid to each well followed by measuring the absorbance at 450nm using a fluorescence plate reader.

Y=((((P1+x+K1)−((−P1−x−K1)̂2−(4*x*P1))̂0.5)/2*(P3−P4)/P1)+P4)   EquationS1:

-   Y=% fluorescence quench proportional to [ARMU2−uPAR] complex-   x=[uPAR] added-   P1=total conc. of ARM-U2-   P3=maximum fluorescence quenching signal-   P4=minimum fluorescence quenching signal-   K1=dissociation constant for ARMU2/uPAR binding

Y=A _(min)+(A _(Max) −A _(Min))/(1+10̂((X−Log IC ₅₀))) where X=conc ofARM-U2[M],Y=fraction of uPAR bound to ARM-U2  Equation S2:

K _(I) =K _(D)1*[complex1]/2*(1/(−[uPA _(T)]+[complex1]/2)−IC ₅₀/([uPA_(T)]*([complex1]/2−[uPAR _(T)])+[complex1]/2*(K_(D)1−[complex1]/2+[uPAR _(T)])))  Equation S3:

-   K_(D)1=K_(D) (uPA−uPAR)=0.2 nM-   K₁=K₁ (ARMU2−uPAR)-   [complex1]=cone of uPAR−uPA complex-   [uPA_(T)]=total cone of uPA=100 nM-   [uPAR_(T)]=total cone of uPAR=10 nM-   IC₅₀ for ARMU2 binding uPAR

Antibody Recruiting Assays

A172 glioblastoma cells (ATCC, CRL-1620) were suspended in Assay Media(phenol-free RPMI 1640 medium plus 10% ultra-low IgG FBS). 10⁵ cellstaken up in 50 μl media or media containing exogenous uPAR were mixedwith a fixed concentration of the indicated ARM-U2 compound from100×DMSO stocks. After a 1 hour incubation on ice, cells were washedwith 1.5 mL of cold Assay Media, pelleted for 2 min at 200 rcf,aspirated, and resuspended in 100 L of fresh assay media with 133 nM ofanti-DNP-biotin-xx conjugate antibody□ (Invitrogen). Cells were allowedto incubate for an additional 30 min on ice, L of a 2 mg/mL stock ofstreptavidin-AlexaFluor647 conjugate□ and then 1 (Invitrogen) was added.Negative control tubes contained streptavidin+/−antibody in the absenceof ARM-U2. After 15 min more on ice, cells were washed 2× with 1.5 mL ofcold Assay Media before flow cytometric analysis. Propidium iodide wasadded from a 1000× stock as a cell viability stain (FL-3 positive) andantibody recruiting to the cell surface was evaluated by measuringincreasing cell counts in the FL-4 channel negative for FL-3 (lowerright quadrant of representative dot plots). The evaluation of selectivebinding of ARM-U2 to uPAR was carried out by performing the antibodyrecruiting assay described above identically, save that B16-F10 melanomacells, either stably transfected with human uPAR or the empty vectorpcDNA3.1 (isogenic negative control), were used in place of A172 cells.The results of these assays is presented in FIGS. 15-17.

Antibody-Dependent Cellular Phagocytosis (ADCP) Assays

IFNγ-primed U937 cells were stained with DiD dye (final concentration1.9 μM) for 30 min at 37° C. A172 target cells were prepared by stainingadherent cells with DiO dye (Invitrogen; final concentration 1.9 μM),and then nonenzymatically detaching the cells with 0.5 mM EDTA and EGTA.Cells were counted with trypan blue staining to verify cell viability.To measure phagocytosis, 2.5×10⁴ target cells were suspended in 25 ulphenol-free low-IgG RPMI media to which 25 ul of 100 nM anti-DNPantibody (rabbit polyclonal KLH IgG, Invitrogen-100× stock) containingmedia was added followed by the addition of 1 ul of ARM-U2 from 100×DMSOstocks or 6-ABT-4 from 100×DMSO stocks for compound out-competitionexperiments, followed by the addition of 50 ul of 10⁵ U937 cells in thesame RPMI Assay Media resulting in a final volume of 100 uL. Thisexperimental setup yielded an effector-to-target ratio (E:T ratios) of4:1. For out-competition studies with exogenous uPAR, 250 ul of A-172target cells in assay media was added directly to lyophilized 100 uguPAR (R@D systems) to yield an 8.4 uM stock uPAR concentration, this wasthen combined in the manner and order described above. Eppendorfs werecentrifuged at 200 rcf for 2 min then incubated at 37° C. for 1 hour orat 4° C. for 1 hr in controls establishing real phagocytosis is indeedrepresented by double positives in flow cytometry. Phagocytosis washalted by placing tubes on ice. Flow cytometric measurements were thenmade using an Accuri C6 flow cytometer with forward scatter and sidescatter gating parameters initially set collecting on live intact cells.The live cell population selected was further gated into four quadrantsusing dual FL-1 and FL-4 fluorescence enabling for the differentiationof effector only cells (top left quadrant-high FL-4), target only cells(bottom right quadrant-high FL-1 fluorescence), and double positiveoverlapping target and effector cells (top right quadrant-high FL-1 andFL-4 fluorescence). The upper right quadrant indicating the number ofphagocytosed target cells was used to calculate the percentphagocytosis: % targets phagocytosed=[(double-positive cells)/(remainingtarget cells+double-positive cells)×100%]−background phagocytosis.Results are shown in FIGS. 18-21.

Inflammatory Cytokine Release ELISA

Analysis of u937 secretion of 12 human inflammatory cytokines in thepresence of anti-DNP antibodies and A172 target cells induced by ARM-U26-ABT-1 was carried out using the Multi-Analyte ELISArray Kit (MEH-004A,Qiagen). Sample preparation involved the immobilization of A172 targetcells (approx. 1,000,000/3 ml T-flask) over the course of 24 hours at37° C. followed by the addition of 1.5 ml phenol free RPMI assay media.30 ul of 6-ABT-1 from 100×DMSO stocks or DMSO only was added (50 nMfinal 6-ABT-1 conc, 1% DMSO) followed by the addition of 30 ul anti-DNPantibody from 100× aqueous stocks (133 nM final concentration). 1.5 mlof IFN-gamma primed U937 cells suspended in phenol free RPMI assay mediaat a concentration of 2666.6 cells/ul, was then added and the plateincubated at 37° C. for 24 hr. Following incubation, the supernatant wascollected, pelleted by centrifugation, and the supernatant collected andstored on ice. The ELISA plate pre-loaded with immobilized antibodiesspecific for each of the 12 cytokines tested was loaded with the cellsupernatant collected, washed, and the bound cytokine detected all asdescribed in the associated instruction manual available from Qiagen.The ELISA plate was read at an absorbance of 450 nm using a Perkin-ElmerFluorescence plate reader with increases in absorbance at 450 nmproportional to bound cytokine. Results are show in FIG. 22.

Amnis Imagestream Imaging

Phagocytosis experiments were conducted as described above for ADCPassays only cellular samples were fixed following 1 h incubation at 37°C. using 3% formaldehyde for 30 min on ice. Cells were washed once inPBS, then stained with anti-CD14-APC and anti-CD11b-APC antibodies(Biolegend) plus Hoechst dye (Invitrogen) for 30 minutes on ice. U937monocytes were also stained with DID while A172 target cells werestained with DIO membrane dyes. The cells were washed once in PBS, thenanalyzed on an Amnis Imagestream X flow cytometer, where images and datafor 30,000 events/sample were collected. Data was analyzed using AmnisIDEAS software.

ADCC Using the xCELLigence System

One day before the experiment, A172 cells were detached, counted,aspirated, and diluted in FBS ADCC media (to a final concentration of25,000 cells/mL). Into each well of an E-plate was added 200 μL of thecell suspension (5,000 cells). The plate was allowed to stand at ambienttemperature (30 min) and maintained in an incubator (37° C., 12 h). ThexCelligence system was maintained inside the incubator (37° C.). AnE-plate containing 100 μL FBS ADCC media per well was used for obtainingbackground measurements. The seeded E-plate was placed in the port, andcell index readings were obtained (every 2 min for 30 min) to confirmthat the cells had adhered properly. The wells were aspirated and toeach well was added 50 μl of FBS-free RPMI media followed by theaddition of 0.15 μl of a 1000× stock of 2 in DMSO followed by theaddition of 1.5 ul of Rabbit IgG anti-DNP from a 2 mg/mL stock to givefinal concentrations of 133 nM antibody and 10 nM, 50 nM, or 1 μM 2. TheE-plate was returned to the port, and cell index readings were obtained(every 2 min for 90 min). A solution of u937 cells in FBS-free RPMImedia as described above for ADCP assays, was prepared and 100 μl addedto each well of the E-plate. The E-plate was returned to the port, andcell index readings were obtained (every 2 min for 24 h, 37° C.). Cellindex readings were normalized (RTCA software) at the time pointimmediately after addition of the u937 cells. Figure S13 shows therepresentative plots of changes in cell index with time in the presenceand absence of ARM-U2, antibody, and competitor compound 20. Values fromthe 24-hour time point were used to calculate specific killing. Normalgrowth was defined as that shown by target A172 cells treated witheffector cells and anti-DNP antibody, but no compound. Specific killingwas calculated as the following formula:

% specific killing=[1−(normalized cell index observed)/(normalgrowth)]*100

Isoxazole Cleavage to Corresponding Compounds Synthetic Procedure:

Upon exposure of a red solution of 10 mg of an ARM-U2 compound (6-ABT-1)in 1 ml DMSO to room light for up to 48 h or longer, a color change topurple was observed. The conversion/degradation occurs much more quicklyin aqueous solution (t_(1/2)≈3 h) relative to DMSO (t_(1/2)≈24 h). TheHPLC purification of this mixture resulted in isolation of 23 as purplesolid obtained in 50% yield (5 mg). ¹H NMR (500 MHz, DMSO-d₆) 12.05 (s,1H), 8.95 (t, J=5.4 Hz, 1H), 8.81 (d, J=2.7 Hz, 1H), 8.25-8.21 (m, 5H),8.08 (s, 1H), 7.88 (brs, 1H) 7.87-7.78 (m, 2H), 7.50 (dd, J=8.2, 2 Hz1H), 7.28 (d, J=8.3 Hz, 1H), 7.20 (d, J=9.7 Hz, 1H), 7.09 (s, 1H),3.55-3.47 (m, 12H), 3.40 (t, J=6.3 Hz, 2H), 3.22-3.08 (m, 2H), 2.96-2.71(brm, 4H), 2.60 (m, 1H), 1.67-1.62 (m, 4H), 1.28-1.22 (n, 4H). HRMS(ES+) calc'd for C₄₂H₄₈N₇O₁₆S₂(M+H proposed structure MW) Exact Mass:970.2521 m/z. Found 970.2534 Additional carbon and two dimensional NMRcharacterization techniques will further confirm proposed structure of23.

Biological Activity of Compound 23

Confirmation of Activity Against uPAR

Evaluation of binding affinity of ARM-U2 degradation product to 23 usinga competitive ELISA. An IC₅₀ of 63 nM was observed translating tocalculated K_(I) of 10 nM demonstrating no change in uPAR bindingaffinity relative to ARM-U2. The results of this assay are presented inFIG. 23. The observation is consistent with literature reports of SARstudies on IPR-803 analogs and our computational and crystallographicdata on ARM-U2 binding to uPAR.

Significance of the Present Invention

The selectivity and high affinity of ARM-U2 for uPAR which isover-expressed on many cancer cell types enables for its potentialapplication as a therapeutic targeting a wide range of cancer celltypes. ARM-U2 employs a dual mode of action as an anticancer therapeuticby disrupting the native uPA uPAR interaction as an antagonist and bytargeting metastatic cancer cells for destruction by the host immunesystem as an antibody recruiting molecule and shows that employing smallmolecules to redirect the cytotoxic functions of antibodies selectivelyagainst cancer cells, might reduce the toxicity associated with othercancer fighting strategies.

1. A compound according to the general formula: Where

is a moiety which binds to an active site of urokinase-type plasminogenactivator receptor (uPAR) on the surface of cancer cells of a patient orsubject;

is an antibody binding moiety comprising a hapten which is capable ofbinding to an antibody in said patient or subject (preferably anendogenous antibody which pre-exists in the patient or subject withouthaving to be raised prior to therapy); Each L1 is a linker moleculewhich chemically links

to CT, L2 or

in said compound; Each L2 is a linker molecule which chemically links

to CT, L1 or

in a molecule; Each CT is independently an optional connector moleculewhich, when present links L1 or L2 to

, L1 or L2 to

and L1 to L2; Each j is independently 0, 1, 2, 3, 4 or 5 (preferably 0or 1, more preferably 1); Each k is independently 0, 1, 2, 3, 4 or 5(preferably 0 or 1, more preferably 1), with the proviso that at leastone CT is present when k and j are both 0 (preferably at least one of kand j is 1); and Each m and n is independently an integer from 1 to 15,1 to 10, 1 to 5, 1 to 3, 2 to 3, 2 to 5, 1 to 2 or 1 (preferably m is 1and n is 1, 2 or 3), or a pharmaceutically acceptable salt, solvate orpolymorph thereof.
 2. A compound of claim 1 according to the generalformula:

Wherein each R^(N) is independently H or a C₃-C₃ alkyl group when N isan amine group or each R^(N) is absent when N forms all isoxazole groupby binding to the adjacent oxygen atom; R₁, R₂, R₃, R₄ and R₅ are eachindependently H, a halogen (F, Cl, Br, I, preferably F), a C₁-C₃ alkylgroup optionally substituted with one or two hydroxyl groups or up tothree fluoro groups, NO₂, CN, a (CH₂)_(m′)OR^(E) (O-alkyl) group, a(CH₂)_(m′)COR^(E) (keto) group, a (CH₂)_(m′)COOR^(E) (carboxy ester)group, a (CH₂)_(m′)SO₃H group, a (CH₂)_(m′)OCOR^(E) (oxycarbonyl ester)group,

Each R′ is independently H or a C₁-C₃ alkyl group (preferably H or CH₃,most often H); R_(a) is a sidechain derived from a natural or unnaturalamino acid (D- or L-, preferably a L-amino acid) preferably selectedfrom the group consisting of alanine (methyl), arginine(propyleneguanidine), asparagine (methylenecarboxyamide), aspartic acid(ethanoic acid), cysteine (thiol, reduced or oxidized di-thiol),glutamine (ethylcarboxyamide), glutamic acid (propanoic acid), histidine(methyleneimidazole), isoleucine (1-methylpropane), leucine(2-methylpropane), lysine (butyleneamine), methionine(ethylmethylthioether), phenylalanine (benzyl), proline (R′ forms acyclic ring with R_(a) and the adjacent nitrogen group to form apyrrolidine group), hydroxyproline, serine (methanol), threonine(ethanol, 1-hydroxyethane), tryptophan (methyleneindole), tyrosine(methylene phenol) or valine (isopropyl) Each R^(E) is H or a C₁-C₆alkyl group optionally substituted with one or two hydroxyl groups or upto three chloro or fluoro groups (preferably R^(E) is H or a C₁-C₃ alkylgroup); R_(1′), R_(2′), R_(3′), R_(4′) and R_(5′) are each independentlyH, a halogen (F, Cl, Br, I, preferably F), a C₁-C₆ (preferably C₁-C₃)alkyl group optionally substituted with one or two hydroxyl groups or upto three chloro or fluoro groups, NO₂, CN, a (CH)_(m′)OR^(E) (O-alkyl)group, a (CH₂)_(m′)COOR^(E) (carboxy ester) group, a (CH₂)_(m′)O—COR^(E)(oxycarbonyl ester) group or a (CH₂)_(m′)COR^(E) (keto) group; Each m′is independently 0, 1, 2, 3, 4, 5, or 6 (preferably 0, 1, 2 or 3, morepreferably 0 or 1); Each y′ is independently 0, 1 or 2 (preferably 0 or1); R^(LABT) is an

 group, where L is a bond, at least one linker (preferably a singlelinker) which comprises a first linker group L1 which optionallyincludes a connector group CT and an optional linker group L2 whichitself optionally includes a connector group CT, said first linker groupL1 being linked to said second linker group L2 optionally (preferably)through a CT group; and

is an antibody binding moiety comprising a hapten which is capable ofbinding to an antibody in said patient or subject, or a pharmaceuticallyacceptable salt, stereoisomer, enantiomer, solvate or polymorph thereof.3. The compound according to claim 1 according to the chemicalstructure:

Where the substituents on the compound R′, R_(1′), R_(2′), R_(3′),R_(4′), R_(5′), R₁, R₂, R₃, R₄, R₅ and R^(LABT) are the same as for thecompound of claim 2, or a pharmaceutically acceptable salt,stereoisomer, enantiomer, solvate or polymorph thereof.
 4. The compoundaccording to claim 1 according to the chemical structure;

Where the substituents R^(N), R′, R_(1′), R_(2′), R_(3′), R_(4′),R_(5′), R₁, R₂, R₃, R₄, R₅ and R^(LABT) are the same as for the compoundof claim 2 above.
 5. The compound according to claim 2 wherein R₁ is H,CO₂H or SO₃H; R₂ is H, CO₂H, SO₃H, —NHCH₂—CO₂H, —NHCH₂—SO₃H,—(O)—NHCH₂—CO₂H or —C(O)—NHCH₂—SO₃H; R₃ is H, CO₂H, SO₃H, —NHCH₂—CO₂H,—NHCH₂—SO₃H, —C(O)—NHCH₂—CO₂H or —C(O)—NHCH₂—SO₃H (preferably H, CO₂H orSO₃H); R₄ is H, SO₃H or CO₂H (preferably H or CO₂H; R5 is H, SO₃H orCO₂H (preferably H) and R′, R_(1′), R_(2′), R_(3′), R_(4′) and R_(5′)are H, or a pharmaceutically acceptable salt, stereoisomer, enantiomer,solvate or polymorph thereof.
 6. The compound according to claim 2wherein R₃ is H, CO₂H or SO₃H; R₄ is H or CO₂H; and R5 is H.
 7. Thecompound according to claim 2 wherein R₁ is H or SO₃H, R₂ is H, CO₂H,SO₃H, —NHCH₂—CO₂H, —NHCH₂—SO₃H, —C(O)—NHCH₂—CO₂H or —C(O)—NHCH₂—SO₃H, R₃is H or SO₃H and R₄ is H or CO₂H, or a pharmaceutically acceptable salt,stereoisomer, enantiomer or solvate thereof.
 8. The compound accordingto claim 2 wherein

is

where X_(L) is N(R¹), O, S, S(O), SO₂, S(O)₂O, —OS(O)₂, or OS(O)₂O; andR¹ is H, a C₁-C₃ alkyl group or a —C(O)(C₁-C₃) group, preferably H; eachn and n′ is independently 1 to 25, 1 to 15, 1 to 12, 2 to 11, 2 to 10, 2to 8, 2 to 6, 2 to 5, 2 to 4 and 2 to 3 or, 2, 3, 4, 5, 6, 7, or 8; andeach n″ is independently 0 to 8, often 1 to 7, or 1, 2, 3, 4, 5 or 6(preferably 3).
 9. The compound claim 2 wherein L is a linker groupbased upon polyethyleneglycol (PEG) linkages, polypropylene glycollinkages, or polyethyleneglycol-co-polypropylene oligomers (up to about100 units, about 1 to 100, about 1 to 75, about 1 to 60, about 1 to 50,about 1 to 35, about 1 to 25, about 1 to 20, about 1 to 15, 2 to 10,about 4 to 12, about 1 to 8, 1 to 3, 1 to 4, 2 to 6, 1 to 5, etc.). 10.The compound according claim 2 wherein L is a linker group according tothe chemical structure:

where each n and n′ is independently 1 to 25, 1 to 15, 1 to 12, 2 to 11,2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4 and 2 to 3 or 1, 2, 3, 4, 5, 6,7, or
 8. 11. The compound according to claim 2 wherein L is a linkergroup according to the chemical structure:

where each n and n′ is independently 1 to 25, 1 to 15, 1 to 12, 2 to 11,2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4 and 2 to 3 or 1, 2, 3, 4, 5, 6,7, or 8; and each n″ is independently 0 to 8, often 1 to 7, or 1, 2, 3,4, 5 or
 6. 12. The compound claim 2 wherein L is a polyamino acidoptionally comprising one or two connector groups CT comprising up to100 (preferably about 1 to 75, about 1 to 60, about 1 to 50, about 1 to45, about 1 to 35, about 1 to 25, about 1 to 20, about 1 to 15, 2 to 10,about 4 to 12, about 5 to 10, about 4 to 6, about 1 to 8, about 1 to 6,about 1 to 5, about 1 to 4, about 1 to 3) amino acid residues whereinsaid amino acid residues are selected from naturally occurring D and Lamino acids or a group according to the chemical structure:

Where R_(a) is H, C₁-C₃ alkyl or alkanol or forms a cyclic ring with R³(proline) and R³ is a side chain derived from an amino acid preferablyselected from the group consisting of alanine (methyl), arginine(propyleneguanidine), asparagine (methylenecarboxyamide), aspartic acid(ethanoic acid), cysteine (thiol, reduced or oxidized di-thiol),glutamine (ethylcarboxyamide), glutamic acid (propanoic acid), glycine(H), histidine (methyleneimidazole), isoleucine (1-methylpropane),leucine (2-methylpropane), lysine (butyleneamine), methionine(ethylmethylthioether), phenylalanine (benzyl), proline (R³ forms acyclic ring with R, and the adjacent nitrogen group to form apyrrolidine group), hydroxyproline, serine (methanol), threonine(ethanol, 1-hydroxyethane), tryptophan (methyleneindole), tyrosine(methylene phenol) or valine (isopropyl); and m (within the context ofthis use) is an integer from 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to50, 1 to 45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to6, 1, 2, 3, 4 or
 5. 13. The compound according to claim 2 wherein L is alinker group according to the chemical formula:

Where Z and Z′ are each independently a bond, —(CH₂)_(i)—O,—(CH₂)_(i)—S, —(CH₂)_(i)—N—R,

wherein said —(CH₂)_(i) group, if present in Z or Z′, is bonded to aconnector (CT), an alternative linker, A_(B)M and/or UPAR_(B)M; Each Ris H, or a C₁-C₃ alkyl or alkanol group; Each R² is independently H or aC₁-C₃ alkyl group; Each Y is independently a bond, O, S or N—R; Each iis independently 0 to 100, 0 to 75, 1 to 60, 1 to 55, 1 to 50, 1 to 45,1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 0, 1, 2, 3,4 or 5; D is

 or a bond, with the proviso that Z, Z′ and D are not eachsimultaneously bonds; j is 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to 50,1 to 45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1,2, 3, 4 or 5; m′ is 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to 50, 1 to45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1, 2,3, 4 or 5; n is 1 to 100, 1 to 75, 1 to 60, to 55, 1 to 50, 1 to 45, 1to 40, 2 to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1, 2, 3, 4 or5 (n is preferably 2); X¹ is O, S or N—R; and R is as described above,or a pharmaceutical salt thereof.
 14. A compound according to claim 2wherein linker group L is a group according to the chemical structure:

or a polypropylene glycol or polypropylene-co-polyethylene glycol linkercontaining between 1 and 100 alkyleneglycol units; Where R_(a) is H,C₁-C₃ alkyl or alkanol or forms a cyclic ring with R³ when R³ is asidechain of proline and R³ is a side chain derived from a naturallyoccurring D- or L-amino acid; Each m is independently an integer from 1to 100; and Each n is independently an integer from 1 to
 100. 15. Acompound according claim 2 wherein said A_(B)M group is a groupaccording to the chemical structure:

Where Y′ is H or NO₂ (preferably H); X is O, CH₂, NR¹, S(O), S(O)₂,—S(O)₂O, —OS(O)₂, or OS(O)₂O; and R¹ is H, a C₁-C₃ alkyl group, or a—C(O)(C₁-C₃) group; and X_(R) is O, S or NR¹.
 16. The compound accordingto claim 2 wherein said A_(B)M group is a group according to thechemical structure:

Where R^(NO2) is a nitrophenyl or a dinitrophenyl group linked throughan amino or thiol group as indicated; or a group according to thechemical structure:

Where Y′ is H; X is O, CH₂, NR¹, S(O), S(O)₂, —S(O)₂O, —OS(O)₂, orOS(O)₂O; and R¹ is H, a C₁-C₃ alkyl group, or a —C(O)C₁-C₃) group. 17.The compound according to claim 2 wherein said A_(B)M group is a groupaccording to the chemical structure:

a group represented by the chemical formula:

Where X′ is CH₂, O, N—R¹, or S, preferably O; R^(1′) is H or C₁-C₃alkyl; and Z is a bond, a monosaccharide, disaccharide, oligosaccharide,glycoprotein or glycolipid.
 18. The compound according to claim 2wherein said A_(B)M group is a group according to the chemicalstructure:

Where X_(R) is O, S or NR¹; and X_(M) is O, NR¹ or S, and R¹ is H or aC₁-C₃ alkyl group.
 19. The compound according to claim 15 wherein saidA_(B)M group comprises from one to four rhamnose groups.
 20. Thecompound according to claim 2 wherein said A_(B)M group is a groupaccording to the chemical structure:

Where X″ is O, CH₂, NR¹, S; and R¹ is H, a C₁-C₃ alkyl group or a—C(O)(C₁-C₃) group; or

Where X^(b) is a bond, O, CH₂ or NR¹ or S; and R¹ is the same as above;or a group according to the chemical structure:

Where DNP is a dinitrophenyl group; or a dinitrophenyl group accordingto the chemical structure:

Where Y′ is H; X is O, CH₂, NR¹, S(O), S(O)₂, —S(O)₂O, —OS(O)₂, orOS(O)₂O; and R¹ is H, a C₁-C₃ alkyl group, or a —C(O)(C₁-C₃) group; or afluorescein group according to the chemical structure:


21. The compound according to claim 2 wherein said A_(B)M group is adinitrophenyl group or a rhamnose group.
 22. (canceled)
 23. The compoundaccording to claim 2 wherein said CT group is a group according to thechemical structure:

or a diamide group according to the structure:

Where X² is CH₂, O, S, NR⁴, C(O), S(O), S(O)₂, —S(O)₂O, —OS(O)₂, orOS(O)₂O; X³ is O, S, NR⁴; R⁴ is H, a C₁-C₃ alkyl or alkanol group, or a—C(O)(C₁-C₃) group; Each R¹ is independently H or a C₁-C₃ alkyl group(preferably H); and n″ is independently 0 to 8, often 1 to 7, or 1, 2,3, 4, 5 or 6 (preferably 3).
 24. The compound according to claim 23wherein said CT group is a group according to the chemical structure:

Where n″ is 1-7.
 25. The compound of claim 2 according to any of thechemical structures of FIG. 4, hereof or a pharmaceutically acceptablesalt, stereoisomer or enantiomer thereof.
 26. The compound of claim 2which is 1-ABT-1, 1-ABT-2, 1-ABT-3, 2-ABT-1, 2-ABT-2, 2-ABT-3, 3-ABT-1,3-ABT-2, 3-ABT-3, 4-ABT-1, 4-ABT-2, 4-BT-3, 5-ABT-1, 5-ABT-2, 5-ABT-3,6-ABT-1, 6-ABT-2 or 6-ABT-3 of FIG. 4 or a pharmaceutically acceptablesalt, stereoisomer or enantiomer thereof.
 27. The compound of claim 2which is 1-ABT-1, 1-ABT-2, 2-ABT-1, 2-ABT-2, 3-ABT-1, 3-ABT-2, 4-ABT-1,4-ABT-2, 5-ABT-1, 5-ABT-2, 6-ABT-1 or 6-ABT-2 of FIG. 4 or apharmaceutically acceptable salt, stereoisomer or enantiomer thereof.28. The compound of claim 2 which is 6-ABT-1 or an enantiomer thereof.29. The compound of claim 1 according to any one of the chemicalstructures of FIG. 7, or a pharmaceutically acceptable salt,stereoisomer or enantiomer thereof.
 30. A pharmaceutical compositioncomprising an effective amount of a compound according to claim 2, incombination with a pharmaceutically acceptable carrier, additive orexcipient.
 31. A pharmaceutical composition according to claim 30further in combination with an additional anticancer agent.
 32. Thecomposition according to claim 31 wherein said additional anticanceragent is an antimetabolite, an inhibitor of topoisomerase I and II, analkylating agent, a microtubule inhibitor or a mixture thereof.
 33. Thecomposition according to claim 31 wherein said additional anticanceragent is a FLT-3 inhibitor, a VEGFR inhibitor, an EGFR TK inhibitor, anaurora kinase inhibitor, a PIK-1 modulator, a Bcl-2 inhibitor, an HDACinhibitor, a c-MET inhibitor, a PARP inhibitor, a Cdk inhibitor, an EGFRTK inhibitor, an IGFR-TK inhibitor, an anti-HGF antibody, a PI3 kinaseinhibitors, an AKT inhibitor, a JAK/STAT inhibitor, a checkpoint-1 or 2inhibitor, a focal adhesion kinase inhibitor, a Map kinase kinase (mekinhibitor, a VEGF trap antibody or a mixture thereof.
 34. Thecomposition according to claim 31 wherein said additional anticanceragent is everolimus, trabectedin, abraxane, TLK 286, AV-299, DN-101,pazopanib, GSK690693, RTA 744, ON 0910.Na, AZD 6244 (ARRY-142886,AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin, vandetanib, ARQ-197,MK-0457, MLN8054, PHA-739358, R-763, AT-9263, pemetrexed, erlotinib,dasatanib, nilotinib, decatanib, paniumumrab, arubicin, oregovomab,Lep-etu, nolatrexed, azd2171, batabulin, ofatumumab, zanolimumab,edotecarin, tetrandrine, rubitecan, tesmilifene, oblimersen,ticilimumab, ipilimumab, gossypol, Bio 111, 131-I-TM-601, ALT-110, BIO140, CC 8490, cilengitide, gimatecan, IL13-PE38QQR, INO 1001, IPdR₁KRX-0402, lucanthone, LY 317615, neuradiab, vitespan, Rta 744, Sdx 102,talampanel, atrasentan, Xr 311, romidepsin, ADS-100380, sunitinib,5-fluorouracil, vorinostat, etoposide, gemcitabine, doxorubicin,liposomal doxorubicin, 5′-deoxy-5-fluorouridine, vincristine,temozolomide, ZK-304709, seliciclib; PD0325901, AZD-6244, capecitabine,L-Glutamic acid,N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-,disodium salt, heptahydrate, camptothecin, PEG-labeled irinotecan,tamoxifen, toremifene citrate, anastrazole, exemestane, letrozole, DES(diethylstilbestrol), estradiol, estrogen, conjugated estrogen,bevacizumab, IMC-1C11, CHIR-258);3-[5-(methylsulfonylpiperadinemethyl)-indolylj-quinolone, vatalanib,AG-013736, AVE-0005, the acetate salt of [D-Ser(But) 6, Azgly10](pyro-Glu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-Azgly-NH₂ acetate[C₅₉H₈₄N₁₈Oi₄-(C₂H₄O₂)_(x) where x=1 to 2.4], goserelin acetate,leuprolide acetate, triptorelin pamoate, medroxyprogesterone acetate,hydroxyprogesterone caproate, megestrol acetate, raloxifene,bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714;TAK-165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF antibody,erbitux, EKB-569, PKI-166, GW-572016, Ionafarnib, BMS-214662,tipifarnib; amifostine, NVP-LAQ824, suberoyl analide hydroxamic acid,valproic acid, trichostatin A, FK-228, SU11248, sorafenib, KRN951,aminoglutethimide, arnsacrine, anagrelide, L-asparaginase, BacillusCalmette-Guerin (BCG) vaccine, bleomycin, buserelin, busulfan,carboplatin, carmustine, chlorambucil, cisplatin, cladribine,clodronate, cyproterone, cytarabine, dacarbazine, dactinomycin,daunorubicin, diethylstilbestrol, epirubicin, fludarabine,fludrocortisone, fluoxymesterone, flutamide, gemcitabine, hydroxyurea,idarubicin, ifosfamide, imatinib, leuprolide, levamisole, lomustine,mechlorethamine, melphalan, 6-mercaptopurine, mesna, methotrexate,mitomycin, mitotane, mitoxantrone, nilutamide, octreotide, oxaliplatin,pamidronate, pentostatin, plicamycin, porfimer, procarbazine,raltitrexed, rituximab, streptozocin, teniposide, testosterone,thalidomide, thioguanine, thiotepa, tretinoin, vindesine,13-cis-retinoic acid, phenylalanine mustard, uracil mustard,estramustine, altretamine, floxuridine, 5-deooxyuridine, cytosinearabinoside, 6-mecaptopurine, deoxycoformycin, calcitriol, valrubicin,mithramycin, vinblastine, vinorelbine, topotecan, razoxin, marimastat,COL-3, neovastat, BMS-275291, squalamine, endostatin, SU5416, SU6668,EMD121974, interleukin-12, IM862, angiostatin, vitaxin, droloxifene,idoxyfene, spironolactone, finasteride, cimitidine, trastuzumab,denileukin diftitox, gefitinib, bortezimib, paclitaxel, cremophor-freepaclitaxel, docetaxel, epithilone B, BMS-247550, BMS-310705,droloxifene, 4-hydroxytamoxifen, pipendoxifene, ERA-923, arzoxifene,fulvestrant, acolbifene, lasofoxifene, idoxifene, TSE-424, HMR-3339,ZK186619, topotecan, PTK787/ZK 222584, VX-745, PD 184352, rapamycin,40-O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP-23573, RAD001,ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646,wortmannin, ZM336372, L-779,450, PEG-filgrastim, darbepoetin,erythropoietin, granulocyte colony-stimulating factor, zolendronate,prednisone, cetuximab, granulocyte macrophage colony-stimulating factor,histrelin, pegylated interferon alfa-2a, interferon alfa-2a, pegylatedinterferon alfa-2b, interferon alfa-2b, azacitidine, PEG-L-asparaginase,lenalidomide, gemtuzumab, hydrocortisone, interleukin-11, dexrazoxane,alemtuzumab, all-transretinoic acid, ketoconazole, interleukin-2,megestrol, immune globulin, nitrogen mustard, methylprednisolone,ibritgumomab tiuxetan, androgens, decitabine, hexamethylmelamine,bexarotene, tositumomab, arsenic trioxide, cortisone, editronate,mitotane, cyclosporine, liposomal daunorubicin, Edwina-asparaginase,strontium 89, casopitant, netupitant, an NK-1 receptor antagonists,palonosetron, aprepitant, diphenhydramine, hydroxyzine, metoclopramide,lorazepam, alprazolam, haloperidol, droperidol, dronabinol,dexamethasone, methylprednisolone, prochlorperazine, granisetron,ondansetron, dolasetron, tropisetron, pegfilgrastim, erythropoietin,epoetin alfa, darbepoetin alfa, ipilimumab, nivolomuab, pembrolizumrab,dabrafenib, trametinib, vemurafenib or a mixture thereof.
 35. A methodof treating cancer in a patient in need comprising administered to saidpatient an effective amount of a composition according to claim
 30. 36.(canceled)
 37. (canceled)
 38. The method according to claim 35 whereinsaid cancer is cancer of the prostate, stomach, colon, rectal, liver,pancreatic, lung, breast, cervix uteri, copus uteri, ovary, testis,bladder, renal, brain/CNS, head and neck or throat, Hodgkin's disease,non-Hodgkin's lymphoma, multiple myeloma, leukemia, melanoma,non-melanoma skin cancer, acute lymphocytic leukemia, acute myelogenousleukemia, Ewing's sarcoma, small cell lung cancer, choriocarcinoma,rhabdomyosarcoma, Wilms' tumor, neuroblastoma, hairy cell leukemia,mouth/pharynx, oesophagus, larynx, kidney cancer or lymphoma.
 39. Themethod according to claim 35 wherein said cancer is a metastatic cancer,recurrent cancer or a drug resistant or multiple drug resistant cancer.40. The method according to claim 35 wherein said cancer is prostatecancer, metastatic prostate cancer or recurrent cancer.
 41. A method ofreducing the likelihood that a cancer in a patient will metastasizecomprising administering to a cancer patient in need an effective amountof a composition according to claim
 30. 42. (canceled)
 43. (canceled)44. The method according to claim 41 wherein said cancer is cancer ofthe prostate, stomach, colon, rectal, liver, pancreatic, lung, breast,cervix uteri, corpus uteri, ovary, testis, bladder, renal, brain/CNS,head and neck or throat, Hodgkin's disease, non-Hodgkin's lymphoma,multiple myeloma, leukemia, melanoma, non-melanoma skin cancer, acutelymphocytic leukemia, acute myelogenous leukemia, Ewing's sarcoma, smallcell lung cancer, choriocarcinoma, rhabdomyosarcoma, Wilms' tumor,neuroblastoma, hairy cell leukemia, mouth/pharynx, oesophagus, larynx,kidney cancer or lymphoma.
 45. The method according to claim 41 whereinsaid cancer is prostate cancer.
 46. (canceled)
 47. (canceled)